Hydroxypyruvate reductase promoter elements and methods of use thereof in plants

ABSTRACT

The invention provides compositions and methods for regulating the expression of nucleotide sequence of interest in a plant. Compositions include a novel nucleotide sequence comprising a promoter for the hydroxypyruvate reductase gene. Methods for expressing a nucleotide sequence of interest in a plant using the promoter sequences disclosed herein is provided. The method comprises stably incorporating into the genome of a plant cell a nucleotide sequence operably linked to the promoter of the present invention and regenerating a stably transformed plant that expresses the nucleodide sequence.

FIELD OF THE INVENTION

The invention relates to the field of plant genetic engineering. Moreparticularly, the invention relates to a hydroxypyuvate reductasepromoter. The invention also relates to heterologous nucleic acidconstructs, vectors, transformation methods, and transgenic plant cells,transgenic plants, and transgenic seeds.

BACKGROUND OF THE INVENTION

Transcriptional gene regulation is a key component to the success of anygenetic manipulation. Promoters are genetic elements that bind thetranscriptional apparatus and typically initiate the transcription of adownstream gene or sequence. The downstream gene or sequence can be aselectable marker, a gene encoding a protein of interest, an antisensenucleic acid sequence or a portion of the forgoing or sequences thatwill form non-translatable mRNA such as hairpin structures.

Promoters useful for genetic engineering can typically be classifiedas 1) constitutive, providing gene expression in any cell, 2) tissuespecific, providing expression in a limited type of cell or duringdevelopmental specific time periods, or 3) inducible, providingexpression in response to a cellular or extracellular stimulus. Whileconstitutive promoters have found widespread use to date, there arecircumstances where it is desirable to restrict or limit expression ofthe heterologous sequence.

It is therefore desirable to develop plant promoters which have specificexpression characteristics and maintain a high level of expression inthose tissues or cells.

SUMMARY OF THE INVENTION

The present invention is based in part upon the discovery ofhydroxypyrnvate reductase nucleic acid sequences, polypeptides andpromoter sequence isolated from Arabidopsis thaliana. The promoters,nucleic acids, polynucleotides, proteins and polypeptides, or fragmentsthereof described herein are collectively referred to as HPR promoters,nucleic acids and polypeptides.

Accordingly, in one aspect, the invention provides an isolated HPRpromoter nucleic acid molecule that includes the sequence of SEQ ID NO:4or 5 or a fragment, homolog, analog or derivative thereof. The HPRpromoter nucleic acid is less than 1000 nucleotide in length.Preferably, the HPR promoter is less than 800, 750, or 600 nucleotidesin length.

The HPR promoter regulates transcription of an operably linkednucleotide sequence of interest. The HPR promoter regulatestranscription constitutively. Alternatively transcription is regulatedins response to a stimulus, such as light or an environmental stress(e.g., drought).

Accordingly, in another aspect, the invention provides nucleic acidconstructs including an HPR promoter (e.g., SEQ ID NO:4 or 5) fragment,homolog, analog or derivative thereof operably linked to a nucleotidesequence encoding a gene. Alternatively, the HPR promoter is operablylinked to a non-translatable mRNA molecule of a gene. A non-translatablemRNA molecule includes for example an antisense nucleic acid, a hairpinRNA or a microRNA. The nucleic acid constructs are referred to herein asHPR promoter-gene constructs. Optionally, HPR promoter-gene constructsinclude a nucleic acid encoding a selectable marker such as kanamycin ora reporter gene such as GUS.

The gene is heterologous. Alternatively, the gene is heterologous. Byheterologous gene it is meant a gene other than the native gene whichthe HPR promoter regulates, i.e., hydroxypyruvate reductase. Theheterologous gene encodes a protein of interest or fragment thereof. Aheterologous gene alters an agronomic trait. An agronomic trait is, forexample, disease resistance, herbicide resistance, environmental stressresistance, enhanced growth or increased yield. The heterologous gene isa plant gene. Alternatively, the heterologous gene is a non-plant genes,e.g., mammalian, bacterial, or insect gene. The heterologous gene is astructural gene such as an enzyme, (e.g., farnesyl transferase alpha,farnesyl transferase beta or CaaX prenyl protease), a chaperonin protein(e.g., HSP or Ras), a scaffolding protein or a transcriptionalregulator.

Also included in the invention are vectors containing one or more of theHPR promoters or HPR promoter-gene constructs described herein, and acell containing the vectors. The cell is a plant cell. The plant cell ismonocotyledonous. Alternatively, the plant cell is dicotyledonous.

The invention is also directed to plants and cells transformed with apromoter or a vector comprising a HPR promoter. Also included in theinvention is the seed,and progeny of the transformed plants or cells.

The invention also includes a method of producing a transgenic plant(e.g., monocot or dicot) by introducing to a plant cell a vectorcontaining a HPR promoter-gene construct of the invention to generate atransgenic cell and regenerating a transgenic plant from the transgeniccell. The transgenic plant expresses the protein of interest.Alternatively, the transgenic plant expresses the protein of interest ata decreased level compared to a wild type (non-transformed plant). Forexample, when a plant cell is transformed with a HPR promoter-geneconstruct containing a nucleic acid encoding for a gene of interest, theresulting transgenic plant has an increased level of expression comparedto a wild-type plant. Similarly, when a plant cell is transformed with aHPR-gene construct containing a nucleic acid encoding a non-translatablemRNA molecule of a gene encoding a protein of interest, the resultingtransgenic plant has a decreased level of expression compared to awild-type plant. In some aspects the transgenic plant has an alteredphenotype such as, but not limited to, increased tolerance to stress,altered senescence profile, increased ABA sensitivity, increased yield,increased productivity and increased biomass compared to a wild typeplant. Stress can include a wide variety of conditions including, butnot limited to, drought, heat, salt, photo-oxidative stress or nutrientdeficiency. The invention also includes the transgenic plant, the seed,and progeny of the transformed plants produced by the methods of theinvention.

The invention further provides a method for expressing a heterologousprotein by providing a cell, e.g., a plant cell containing a HPRpromoter-gene construct, a vector that includes a HPR promoter-geneconstruct, and culturing the cell under conditions sufficient to expressthe heterologous protein encoded by the nucleic acid. The expressedprotein is then recovered from the cell. The plant cell ismonocotyledonous. Alternatively, the plant cell is dicotyledonous.Further, a plant cell containing the HPR promoter-gene construct isregenerated to produce a transgenic plant, the transgenic plantexpressing the heterologous protein. The heterologous protein may bepurified from the plant tissue or alternatively the heterologous proteinmay have a biological activity and impart a phenotype to the transgenicplant.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is a schematic representation of the HPR vector constructs; A)pRD29A-anti-FTA, B) pBI121-HPR, C) pRD29A-HPR, D) pBI121-hp-HPR.

FIG. 2. is a schematic representation of the HPR promoter vectorconstructs; A) pHPR-GUS, B) pHPRT-GUS.

FIG. 3. is a illustration of control and transgenic Arabidopsis plantsstained for GUS activity, Row A contains Wild-type and 35S-GUStransgenic seedlings; Row B contains HPR-GUS and pHPRT-GUS transgenicseedlings and Row C contains pHPR-GUS and pHPRT-GUS transgeneic roots

FIG. 4. is an illustration of GUS activity in control and pHPR-GUStransgenic Brassica napus. Panel A contains a wild type seedling andfour transgenic seedlings. Panel B contains siliques and developingembryos from a wild type and transgenic plant.

FIG. 5. is an illustration of GUS activity in controls and transformedcorn callus tissues. Row 1 contains corn callus transformed with thepHPR-GUS construct. Well 2A contains corn callus tissue exposed toAgrobacterium lacking the construct and no transformation protocol. Well2B contains corn callus exposed to Agrobacterium lacking the pHPR-GUSconstruct and exposed to the transformation protocol. Well 3A containsAgrobacterium culture but no corn callus tissue.

DETAILED DESCRIPTION OF INVENTION

The present invention provides structural DNA sequences and theirpromoters, which are active in the tissue of both monocotyledonous anddicotyledonous plants, in particular, the aerial tissue. The presentinvention is based in part on the discovery of a novel promoter sequence(SEQ ID NO:4), of the hydroxyxpyruvate reductase (HPR) gene isolatedfrom Arabidopsis thaliana (At). Also provided is a truncated variationof the promoter sequence. (SEQ ID NO:5). The promoter sequences-arecollectively referred to herein as “HPR promoters”, or “promoters” TheHPR promoters produce high levels of gene expression in Arabidopsis,Brassica (canola) and Zea maise (corn). Northern analysis and GUSreporter activity demonstrate expression in the aerial tissues.Expression is inducible in all shoot tissue, with minimal rootexpression, by light and in response to environmental stress, such asdrought stress. The HPR promoters herein are suitable for applicationsthat require strong expression specifically in aerial tissue.Additionally, if desired the HPR promoter can be modified to includesome root expression. (SEQ ID NO:5)

The invention further provides the hydroxpyruvate reductase (HPR)nucleic acid sequence (SEQ ID NO: 1) and the encoded polypeptide: SEQ IDNO:2, isolated from Arabidopsis thaliana (At) and HPR antisense nucleicacids. (SEQ ID NO:3). The sequences are collectively referred to as “HPRnucleic acids”, HPR polynucleotides” or “HPR antisense nucleic acids”and the corresponding encoded polypeptide is referred to as a “HPRpolypeptide” or “HPR protein”.

Unless indicated otherwise, “HPR” is meant to refer to any of thesequences diclosed herein. Table A below summarizes the nucleic acidsand polypeptides according to the invention TABLE A SEQ ID NAME NTSequence 1 HPR NT+ 2 HPR AA 3 HPR NT− 4 HPR Promoter 5 HPR PromoterTruncated 6 HPRClal AAATCGATATGGCGAAACCGGTGTCC 7 HPRBamHICGGGATCCTCATAGCTTCGAAACAGGCAA 8 HPRBamFW AAAGGATCCATGGCGAAACCGGTGTCCAT 9RD29AP1 TTTAAGCTTGGAGCCATAGATGCAATTCAA 10 RD29AP2AAATCTAGACTTTCCAATAGAAGTAATCAAACC 11 HPRXbaREAAATCTAGACGTTTCCATGTCACAGGTTG 12 HPRSacFW AAAGAGCTCATGGCGAAACCGGTGTCCAT13 HPRSacRE AAAGAGCTCCGTTTCCATGTCACAGGTTG 14 HPRP1HindAAAAAGCTTGAAGCAGCAGAAGCCTTGAT 15 HPR2Bam AAAGGATCCCGCCATGGTAGAGAAAAGAGA16 HPR3Hind AAAAAGCTTACGTCAGCATTATCTCGTTAC 17 Adapter 1CTAATACGACTCACTATAGGGCTCGAGCGGC CGCCCGGGCAGGT 18 Adapter 2 ACCTGCCC-NH219 AP1 GGATCCTAATACGACTCACTATAGGGC 20 28w1 AGCTGGCGTAATAGCGAAGA 21 AP2CTATAGGGCTCGAGCGGC 22 28w2 CGTTGGAGTCCACGTTCTTT 23 28LAP1GTTACTGCTGTGTTTCTTGCGAGGTGACTC 24 28LAP2CTCAAAGCTGAGAACAGAGTCTCTCCCCAATC 25 NPT 1 ATTGCACGCAGGTTCTCCGG 26 NPT 2ATCGGGAGCGGCGATACCG 27 GG9 CTGCATCCGGCGACCTTGTTC 28 HPRXbaFWAAATCTAGAATGGCGAAACCGGTGTCC 29 HPRSaIRV AAAGTCGACTCATAGCTTCGAAACAGGC

Hydroxypyruvate reductase is a component of the photorespiratorypathway. The photorespiratory pathway is a system which evolves carbondioxide and consumes oxygen. Ribisco can react with O₂ resulting insynthesis of 3-phosphoglycerate (3-PGA) and 2-phosphoglycolate. Twomolecules of 2-phosphoglycolate are converted to one CO₂ and onemolecule of 3-PGA thereby salvaging 3 molecules of carbon at the cost ofone molecule of carbon as CO₂. The pathways of phosphoglycolatemetabolism involves three organelles, chloroplast, peroxisome andmitochondrion and the integration of photorespiratory carbon andnitrogen cycles. HPR is localized exclusively in the peroxisome andfunctions in the last step of photorespiration pathway, convertinghydroxypyruvate to glycerate and NADH to NAD. The glycerate is shuttledinto chloroplast, and subsequently converted into glycerate-3-phosphate,which enters the Benson-Calvin cycle for production of carbon skeletons.Recent isotope-labeled feeding studies have firmly established thatchloroplast is the site for ABA biosynthesis, and that pyrnvate andglyceraldehyde 3-phosphate are the ultimate precusors of ABA. One ofthese experiments in which pyruvate and other carbohydrate moleculeswere incubated with intact spinach chloroplasts confirms that pyruvateis incorporated more efficiently into ABA than other compounds such asmevalonic acid (MVA). It is now known that the first step of ABAbiosynthesis is the formation of the C₅ isoprene isopentenyl diphosphate(IPP) from pyruvate and glyceraldyhyde 3-phsophate. The C₅ isoprene isthen converted to β-carotene which is subsequently converted to ABAthrough multiple steps.

The integration of the organelles and the shuttling of intermediatesbetween them provides for the glycerate, formed in the peroxisome, to betransported to the chloroplast. In the chloroplast, conversion to3-phosphoglycerate occurs by glycerate kinase. The fate of the3-phosphoglycerate will be determined by the affinity of various enzymesfor this substrate. For example, chloroplasts also contain a glycolyticpathway catalyzed by plastid specific glycolytic enzymes. This pathwayprovides a means of converting the 3-phosphoglycerate to pyruvate forABA biosynthesis, also a chloroplast localized process. Hydroxypyruvatereductase activity and mRNA transcription is regulated in part by theplant hormone cytokinin and light. (Greenler et al. 1989) Cytokinins area group of plant hormones that have influence on numerous cellularprocesses such as cell division, chloroplast development, celldifferentiation and some enzyme activity. HPR expression and activityare detectable in both light and dark grown seedlings however, both areinduced in cotyledon (Hondred et al. 1987) and leaf (Greenler and Becker1990) in a light dependent manner.

Plant cDNAs have been identified encoding the HPR gene from cucumber(Greenler et al. 1989) and pumpkin (Hayashi et al. 1996). The promoteradjacent to the cucumber gene has also been isolated and characterizedby deletion analysis (Sloan et al. 1993, Daniel and Becker 1995). Thecucumber HPR promoter was analyzed in transgenic tobacco and wasdetermined to require at least 299 bp of 5′-HPR-flanking region toproduce high-level light-regulated expression (Sloan et al. 1993).Continued analysis identified the regions necessary for lightregulation, leaf-specific expression and a negative root elementresponsible for silencing root expression (Daniel and Becker 1995). Inaddition, a cytokinin response element from the cucumber promoter hasalso been mapped (Jin et al. 1998). In a BLAST analysis, it wasdetermined that the cucumber HPR promoter shares no sequence homology,with the HPR promoter of the present invention that was isolated fromArabidopsis. Specifically, the Arabidopsis sequence is half the lengthof the corresponding cucumber sequence.

In a BLAST search of public sequence databases, it was found that thedisclosed 1161 nucleotide Arabidopsis thaliana HRP nucleic acid sequence(SEQ ID NO: 1) which encodes a HPR amino acid sequence (SEQ ID NO;2) is99.8% identical to a portion of the 1428 nucleotide Arabidopsis thalianahydroxypyruvate reductase sequence of Genbank Accession Number D85339and is identical to a portion of the nucleotide Arabidopsis thaliana HRPsequence of Genbank Accession Number AC012563.

The compositions of the invention are useful in regulating theexpression of an operably linked nucleotide sequence of interest. Thepresent invention further provides nucleotide constructs that allowinitiation of transcription in both a constitutive and an induciblemanner. For example, transcription of the operably linked nucleotidesequence is initiated or increased in the presence of a stimulus. Astimulus includes, light, a pathogen, tissue wounding, such as woundingresulting from insect herbivory, leaf breakage by physical means,hormone or chemical exposure, particularly hormones or chemicalsassociated with wounding (i.e., wound-responsive chemicals) or anenvironmental stress such as chilling stress, salt stress, and waterstress.

Additional utilities for HPR promoters, HPR nucleic acids andpolypeptides according to the invention are disclosed herein.

HPR Promoters and Promoter Constructs

The invention provides previously unidentified promoter nucleic acidsequences isolated from the Arabidopsis thaliana (At) HPR gene. The term“promoter” refers to a region of DNA upstream from the translationalstart codon which is involved in recognition and binding of RNApolymerase and other proteins to initiate transcription.

The HPR promoter sequence of the invention includes the nucleic acidsequence of SEQ ID NO:4. The HPR promoter sequences of the invention aretypically identical to or show substantial sequence identity nucleicacid sequence depicted in SEQ ID NO:4. or fragments thereof. A HPRpromoter sequence is at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% 99 identical to SEQ ID NO: 4.

The HPR promoter sequence is 512 nucleotides in length. All or part(i.e., fragment) of the HPR promoter may be used to specifically directexpression of a sequence or gene to plant tissue. Optionally, the HPRpromoter contains additional nucleic acid sequences at the 5′ or 3′ end.The additional nucleic acid sequence is a coding sequence.Alternatively, the additional nucleic acid sequence is a non-codingsequence. For example, the promoter includes other transcriptional andtranslational regulatory nucleic acid sequences (also termed “controlsequences”) that are necessary to express a given gene. In general, thetranscriptional and translational regulatory sequences include, but arenot limited to, promoter sequences, ribosomal binding sites,transcriptional start and stop sequences, translational start and stopsequences, and enhancer or activator sequences. A HPR promoter sequenceis less than 1000 nucleotides in length, e.g., less than or equal to800, 750, 600, 625, 600, 550; 525 nucleotides in length. In variousaspects, the HPR promoter includes the nucleic acid sequence of one ormore of SEQ ID NOs: 4 or 5.

The HPR promoters are capable of conferring high levels (i.e., strongpromoter) of transcription in plant tissue when used as a promoter for aheterologous coding sequence. As used herein, “promoter strength” refersto the level of promoter-regulated expression of a heterologous gene ina plant tissue or tissues, relative to a standard (a standard genepromoter, e.g., the 35S CaMV promoter or the CsVMV promoter). Expressionlevels is measured by linking the promoter to a suitable reporter genesuch as GUS (beta.-glucuronidase). Expression of the reporter gene canbe easily measured by fluorometric, spectrophotometric or histochemicalassays.

Various modifications are made to the promoters of the invention toprovide promoters with different properties (e.g., tissue specificity,promoter strength, and the like). For example, truncated forms of a HPRpromoter are constructed by mapping restriction enzyme sites in thepromoter and then using the constructed map to determine appropriaterestriction enzyme cleavage to excise a subset of the sequence. Themodified promoters are then inserted into a suitable vector and testedfor their ability to drive expression of a marker gene. Tissuespecificity of the modified promoters is tested in regenerated plants.An exemplary modified HPR promoter includes the nucleic acid of SEQ IDNO:5 which allows for expression of a gene of interest in the roottissue of a plant.

HPR promoters are isolated in a variety of ways know in the art. Forexample, HPR promoters are isolated from genomic DNA fragments encodinga HPR protein and which also contain sequences upstream from thesequence encoding the HPR protein. Genomic fragments encoding HPRproteins are isolated by methods known in the art. HPR promotersequences are isolated by screening plant DNA libraries witholigonucleotide probes having sequences derived from the DNA sequence ofthe HPR promoter of SEQ ID NO:4. Other methods known to those of skillin the art can also be used to isolate plant DNA fragments containingHPR promoters. See Sambrook, et al. for a description of othertechniques for the isolation of DNAs related to DNA molecules of knownsequence. For instance, deletion analysis and a promoterless reportergene (e.g., GUS) can be used to identify those regions which can driveexpression of a structural gene. Sequences characteristic of promotersequences can also be used to identify the promoter. Sequencescontrolling eukaryotic gene expression have been extensively studied.For instance, promoter sequence elements include the TATA box consensussequence (TATAAT), which is usually 20 to 30 base pairs upstream of thetranscription start site. In most instances the TATA box is required foraccurate transcription initiation. In plants, further upstream from theTATA box, at positions −80 to −100, there is typically a promoterelement with a series of adenines surrounding the trinucleotide G (or T)N G. J. Messing et al., in Genetic Engineering in Plants, pp. 221-227(Kosage, Meredith and Hollaender, eds. 1983).

The HPR promoter is useful in ligating or fusing (i.e., operably linked)to the 5′ end of one or more nucleic acid sequences (e.g., gene) therebyproducing a HPR promoter--gene construct. The term “operably linked” asused herein refers-to linkage of a promoter upstream from a DNA sequencesuch that the promoter mediates transcription of the DNA sequence. TheHPR promoter is ligated in frame upstream of a sequence to be expressed.Downstream or 3′ of the sequence to be expressed may be suitabletranscription termination signals, including a polyadenylation signal orother sequences found helpful in the processing of the 3′ mRNA terminus.The promoter sequence also includes transcribed sequences between thetranscriptional start and the translational start codon. Optionally, theconstruct contains a nucleic acid encoding a reporter gene or aselectable marker ligated 3′ of the HPR promoter. The reporter/markersequence provides a means to easily identify the cells expressing thesequences under control of the HPR promoter. For example, selectablemarker genes encode a polypeptide that permits selection of transformedplant cells containing the gene by rendering the cells resistant to anamount of an antibiotic that would be toxic to non-transformed plantcells. Selectable marker genes include the neomycin phosphotransferase(nptII) resistance gene, hygromycin phosphotransferase (hpt),bromoxynil-specific nitrilase (bxn), phosphinothricin acetyltransferaseenzyme (B3AR) and the spectinomycin resistance gene (spt), wherein theselective agent is kanamycin, hygromycin, geneticin, the herbicideglufosinate-ammonium (“Basta”) or spectinomycin, respectively.

The nucleic acid sequences is heterologous (i.e., exogenous) to thepromoter. Exogenous and heterologous, as used herein, denote a nucleicacid sequence which is not obtained from and would not normally form apart of the genetic make-up of the plant or the cell to be transformed,in its untransformed state. Foreign genes and sequences, for purposes ofthe present invention, are those which are not naturally occurring inthe plant into which they are delivered. Portions of the above mentionedheterologous or exogenous sequences and foreign genes and sequences areof plant origin, however, the HPR promoter-gene construct forms acombination or variant not naturally occurring in the plant The nucleicacid encodes for a protein of interest or fragment thereof. The gene isfor example a structural gene, an enzyme (e.g., farnesyl transferase,alpha or beta or CaaX prenyl protease), a chaperonin protein (e.g., HSPor Ras)), a scaffolding protein, or a transcriptional regulator. Forexample, the nucleic acid encodes for a gene capable of altering anagronomic trait such as disease resistance, herbicide resistance,environmental stress resistance or increased yield. Alteration ofprenylation by increasing or decreasing farnesyl transferase, CaaXprenyl protease activity has been shown to elicit plants with alteredagronomic traits. (See for example, PCT US 98/15664, U.S. Ser. No.03/26894, WO 02/097097 and WO 03/012116, each of which are incorporatedby reference in their entireties)

The nucleic acid sequences are DNA, such as cDNA and genomic DNA or RNA,such as mRNA and tRNA. For example, the nucleic acid sequence is anon-translated mRNA molecule of a gene or fragment thereof that encodesa protein of interest. Non-translated mRNA includes, e.g., antisense,hairpin RNA, microRNA, or ribozymes. The non-translated mRNA may alteragronomic traits, including those identified above. Alternatively, thenon-translated mRNA may prevent the translation of sequences which aredetrimental to the plant.

The HPR promoter-gene contains one promoter nucleic acid sequence.Alternatively, the HPR promoter-gene construct contains 2, 3, 4, 5, ormore promoter nucleic acid sequences. Optionally, the promoter sequencesare linked together by a spacer. No particular length is implied by theterm spacer. The spacer is less than 1000 nucleotides in length, e.g.,less than or equal to 900, 800, 700, 500, 250, 100, 75, 50, 35, 25, or10 nucleotides in length.

The promoter regulates expression of the nucleic acid sequence ofinterest constitutively. Alternatively, the promoter is inducible by astimulus such as light or an environmental stress, such as drought,chilling stress, salt stress, a pathogen, a herbicide, or wounding. Theterms “constitutive promoter ” as used herein refer to a promoter whichis capable of expressing operably linked DNA sequences in all tissues ornearly all tissues of a plant. The terms “inducible promoter”, as usedherein, refer to plant promoters that are capable of selectivelyexpressing operably linked DNA sequences at particular times in responseto endogenous or external stimuli.

Also included in the invention are vectors containing the HPRpromoter-gene constructs. Suitable plant expression vectors systemsinclude tumor inducing (Ti) plasmid or portion thereof found inAgrobacterium, cauliflower mosaic virus (CaMV) DNA and vectors such aspBI121 .

For expression of HPR promoter gene construct in plants, the recombinantexpression cassette will contain in addition to the HPR promoter andnucleic acid of interest, a transcription initiation site (if the codingsequence to transcribed lacks one), and a transcriptiontermination/polyadenylation sequence. The termination/polyadenylationregion may be obtained from the same gene as the promoter sequence ormay be obtained from different genes. Unique restriction enzyme sites atthe 5′ and 3′ ends of the cassette are typically included to allow foreasy insertion into a pre-existing vector.

Additional regulatory elements that may be connected to the HPR promotergene construct for expression in plant cells include terminators,polyadenylation sequences, and nucleic acid sequences encoding signalpeptides that permit localization within a plant cell or secretion ofthe protein from the cell. Plant signal sequences, including, but notlimited to, signal-peptide encoding DNA/RNA sequences which targetproteins to the extracellular matrix of the plant cell (Dratewka-Kos, etal., J. Biol. Chem., 264: 4896-4900 (1989)) and the Nicotianaplumbaginifolia extension gene (DeLoose, et al., Gene, 99: 95-100(1991)), or signal peptides which target proteins to the vacuole likethe sweet potato sporamin gene (Matsuka, et al., Proc. Nat'l Acad. Sci.(USA), 88: 834 (1991)) and the barley lectin gene (Wilkins, et al.,Plant Cell, 2: 301-313 (1990)), or signals which cause proteins to besecreted such as that of PRIb (Lind, et al., Plant Mol. Biol., 18: 47-53(1992)), or those which target proteins to the plastids such as that ofrapeseed enoyl-ACP reductase (Verwaert, et al., Plant Mol. Biol., 26:189-202 (1994)) are useful in the invention.

A number of types of cells may act as suitable host cells for expressionof the vectors. Plant host cells include cells from monocots and dicots.For example, plant cells include epidermal cells, mesophyll and otherground tissues, and vascular tissues in leaves, stems, floral organs,and roots from a variety of plant species, such as Arabidopsisthlaliana, Nicotiana tabacum, Brassica napus, Zea mays, Oryza sativa,Gossypium hirsutum and Glycine max.

HPR Nucleic Acids

The nucleic acids of the invention include those that encode a HPRpolypeptide or protein. As used herein, the terms polypeptide andprotein are interchangeable.

In some embodiments, a HPR nucleic acid encodes a mature HPRpolypeptide. As used herein, a “mature” form of a polypeptide or proteindescribed herein relates to the product of a naturally occurringpolypeptide or precursor form or proprotein. The naturally occurringpolypeptide, precursor proprotein includes, by way of nonlimitingexample, the full length gene product, encoded by the correspondinggene. Alternatively, it may be defined as the polypeptide, precursorproprotein encoded by an open reading frame described herein. Theproduct “mature” form arises, again by way of nonlimiting example, as aresult of one or more naturally occurring processing steps that may takeplace within the cell in which the gene product arises. Examples of suchprocessing steps leading to a “mature” form of a polypeptide or proteininclude the cleavage of the N-terminal methionine residue encoded by theinitiation codon of an open reading frame, or the proteolytic cleavageof a signal peptide or leader sequence. Thus a mature form arising froma precursor polypeptide or protein that has residues 1 to N, whereresidue 1 is the N-terminal methionine, would have residues 2 through Nremaining after removal of the N-terminal methionine. Alternatively, amature form arising from a precursor polypeptide or protein havingresidues 1 to N, in which an N-terminal signal sequence from residue 1to residue M is cleaved, would have the residues from residue M+1 toresidue N remaining. Further as used herein, a “mature” form of apolypeptide or protein may arise from a step of post-translationalmodification other than a proteolytic cleavage event. Such additionalprocesses include, by way of non-limiting example, glycosylation,myristoylation or phosphorylation. In general, a mature polypeptide orprotein may result from the operation of only one of these processes, ora combination of any of them.

Among the HPR nucleic acids is the nucleic acid whose sequence isprovided in SEQ ID NO:1 or a fragment thereof. Additionally, theinvention includes mutant or variant nucleic acids of SEQ ID NO:1 or afragment thereof, any of whose bases may be changed from thecorresponding base shown in SEQ ID NO:1 while still encoding a proteinthat maintains at least one of its HPR-like activities and physiologicalfunctions. The invention further includes the complement of the nucleicacid sequence of SEQ ID NO:1 including fragments, derivatives, analogsand homologs thereof. Complement nucleic acid HPR sequences include SEQID NO:3. The invention additionally includes nucleic acids or nucleicacid fragments, or complements thereto, whose structures includechemical modifications.

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode HPR proteins or biologically active portions thereof Alsoincluded are nucleic acid fragments sufficient for use as hybridizationprobes to identify HPR-encoding nucleic acids (e.g., HPR mRNA) andfragments for use as polymerase chain reaction (PCR) primers for theamplification or mutation of HPR nucleic acid molecules. As used herein,the term “nucleic acid molecule” is intended to include DNA molecules(e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of theDNA or RNA generated using nucleotide analogs, and derivatives,fragments and homologs thereof. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

“Probes” refer to nucleic acid sequences of variable length, preferablybetween at least about 10 nucleotides (nt), 100 nt, or as many as about,e.g., 6,000 nt, depending on use. Probes are used in the detection ofidentical, similar, or complementary nucleic acid sequences. Longerlength probes are usually obtained from a natural or recombinant source,are highly specific and much slower to hybridize than oligomers. Probesmay be single- or double-stranded and designed to have specificity inPCR, membrane-based hybridization technologies, or ELISA-liketechnologies.

An “isolated” nucleic acid molecule is one that is separated from othernucleic acid molecules that are present in the natural source of thenucleic acid. Examples of isolated nucleic acid molecules include, butare not limited to, recombinant DNA molecules contained in a vector,recombinant DNA molecules maintained in a heterologous host cell,partially or substantially purified nucleic acid molecules, andsynthetic DNA or RNA molecules. Preferably, an “isolated” nucleic acidis free of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated HPR nucleic acid moleculecan contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb or 0.1 kb of nucleotide sequences which naturally flank thenucleic acid molecule in genomic DNA of the cell from which the nucleicacid is derived. Moreover, an “isolated” nucleic acid molecule, such asa cDNA molecule, can be substantially free of other cellular material orculture medium when produced by recombinant techniques, or of chemicalprecursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, a complement ofany of this nucleotide sequence, can be isolated using standardmolecular biology techniques and the sequence information providedherein. Using all or a portion of the nucleic acid sequence of SEQ IDNO:1 as a hybridization probe, HPR nucleic acid sequences or itspromoter can be isolated using standard hybridization and cloningtechniques (e.g., as described in Sambrook et al., eds., MOLECULARCLONING: A LABORATORY MANUAL 2^(nd) Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., eds.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York,N.Y., 1993.)

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to HPR nucleotide sequencescan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

As used herein, the term “oligonucleotide” refers to a series of linkednucleotide residues, which oligonucleotide has a sufficient number ofnucleotide bases to be used in a PCR reaction. A short oligonucleotidesequence may be based on, or designed from, a genomic or cDNA sequenceand is used to amplify, confirm or reveal the presence of an identical,similar or complementary DNA or RNA in a particular cell or tissue.Oligonucleotides comprise portions of a nucleic acid sequence havingabout 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 ntin length. In one embodiment, an oligonucleotide comprising a nucleicacid molecule less than 100 nt in length would further comprise at lease6 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:4 or a complementthereof Oligonucleotides may be chemically synthesized and may be usedas probes.

In another embodiment, an isolated nucleic acid molecule of theinvention includes a nucleic acid molecule that is a complement of thenucleotide sequence shown in SEQ ID NO:1. In another embodiment, anisolated nucleic acid molecule of the invention comprises a nucleic acidmolecule that is a complement of the nucleotide sequence shown in SEQ IDNO:1 or a portion of these nucleotide sequence. A nucleic acid moleculethat is complementary to the nucleotide sequence shown in SEQ ID NO:1 isone that is sufficiently complementary to the nucleotide sequence shownin SEQ ID NO:1 that it can hydrogen bond with little or no mismatches tothe nucleotide sequence shown in SEQ ID NO:1, thereby forming a stableduplex. Exemplary complement nucleic acid sequences to SEQ ID NO:1include the sequences of SEQ ID NO:3.

As used herein, the term “complementary” refers to Watson-Crick orHoogsteen base pairing between nucleotide units of a nucleic acidmolecule, and the term “binding” means the physical or chemicalinteraction between two polypeptides or compounds or associatedpolypeptides or compounds or combinations thereof. Binding includesionic, non-ionic, Von der Waals, hydrophobic interactions, etc. Aphysical interaction can be either direct or indirect. Indirectinteractions may be through or due to the effects of another polypeptideor compound. Direct binding refers to interactions that do not takeplace through, or due to, the effect of another polypeptide or compound,but instead are without other substantial chemical intermediates.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1 e.g., a fragmentthat can be used as a probe or primer, or a fragment encoding abiologically active portion of HPR or a natural promoter of HPR.Fragments provided herein are defined as sequences of at least 6(contiguous) nucleic acids or at least 4 (contiguous) amino acids, alength sufficient to allow for specific hybridization in the case ofnucleic acids or for specific recognition of an epitope in the case ofamino acids, respectively, and are at most some portion less than a fulllength sequence. Fragments may be derived from any contiguous portion ofa nucleic acid or amino acid sequence of choice. Derivatives are nucleicacid sequences or amino acid sequences formed from the native compoundseither directly or by modification or partial substitution. Analogs arenucleic acid sequences or amino acid sequences that have a structuresimilar to, but not identical to, the native compound but differs fromit in respect to certain components or side chains. Analogs may besynthetic or from a different evolutionary origin and may have a similaror opposite metabolic activity compared to wild type.

Derivatives and analogs may be full length or other than full length, ifthe derivative or analog contains a modified nucleic acid or amino acid,as described below. Derivatives or analogs of the nucleic acids orproteins of the invention include, but are not limited to, moleculescomprising regions that are substantially homologous to the nucleicacids or proteins of the invention, in various embodiments, by at leastabout 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (with apreferred identity of 80-99%) over a nucleic acid or amino acid sequenceof identical size or when compared to an aligned sequence in which thealignment is done by a computer homology program known in the art, orwhose encoding nucleic acid is capable of hybridizing to the complementof a sequence encoding the aforementioned proteins under stringent,moderately stringent, or low stringent conditions. See e.g. Ausubel, etal., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NewYork, N.Y., 1993, and below. An exemplary program is the Gap program(Wisconsin Sequence Analysis Package, Version 8 for UNIX, GeneticsComputer Group, University Research Park, Madison, Wis.) using thedefault settings, which uses the algorithm of Smith and Waterman (Adv.Appl. Math., 1981, 2: 482-489, which is incorporated herein by referencein its entirety). A “homologous nucleic acid sequence” or “homologousamino acid sequence,” or variations thereof, refer to sequencescharacterized by a homology at the nucleotide level or amino acid levelas discussed above. Homologous nucleotide sequences encode thosesequences coding for isoforms of a HPR polypeptide. Isoforms can beexpressed in different tissues of the same organism as a result of, forexample, alternative splicing of RNA. Alternatively, isoforms can beencoded by different genes. Homologous nucleotide sequences alsoinclude, but are not limited to, naturally occurring allelic variationsand mutations of the nucleotide sequences set forth herein. Homologousnucleic acid sequences include those nucleic acid sequences that encodeconservative amino acid substitutions (see below) in SEQ ID NO:2 as wellas a polypeptide having HPR activity, e.g. substrate binding.

The nucleotide sequence determined from the cloning of the Arabidopsisthaliana HPR gene allows for the generation of probes and primersdesigned for use in identifying and/or cloning HPR homologues in othercell types, e.g., from other tissues, as well as HPR homologues fromother plants. The probe/primer typically comprises a substantiallypurified oligonucleotide. The oligonucleotide typically comprises aregion of nucleotide sequence that hybridizes under stringent conditionsto at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 ormore consecutive sense strand nucleotide sequence of SEQ ID NO:1; or ananti-sense strand nucleotide sequence of SEQ ID NO:1; or of a naturallyoccurring mutant of SEQ ID NO:1.

Probes based on the Arabidopsis thaliana HPR nucleotide sequence can beused to detect transcripts or genomic sequences encoding the same orhomologous proteins. In various embodiments, the probe further comprisesa label group attached thereto, e.g., the label group can be aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used as a part of a diagnostic test kit foridentifying cells or tissue which misexpress a HPR protein, such as bymeasuring a level of a HPR-encoding nucleic acid in a sample of cellsfrom a subject e.g., detecting HPR mRNA levels or determining whether agenomic HPR gene has been mutated or deleted.

A “polypeptide having a biologically active portion of HPR” refers topolypeptides exhibiting activity similar, but not necessarily identicalto, an activity of a polypeptide of the present invention, includingmature forms, as measured in a particular biological assay, with orwithout dose dependency. A nucleic acid fragment encoding a“biologically active portion of HPR” can be prepared by isolating aportion of SEQ ID NO:1 that encodes a polypeptide having a HPRbiological activity (biological activities of the HPR proteins aredescribed below), expressing the encoded portion of HPR protein (e.g.,by recombinant expression in vitro) and assessing the activity of theencoded portion of HPR. In another embodiment, a nucleic acid fragmentencoding a biologically active portion of HPR includes one or moreregions.

Antisense HPR Nucleic Acids

Another aspect of the invention pertains to isolated antisense nucleicacid molecules that are hybridizable to or complementary to the nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO:1 orfragments, analogs or derivatives thereof. An “antisense” nucleic acidcomprises a nucleotide sequence that is complementary to a “sense”nucleic acid encoding a protein, e.g. complementary to the coding strandof a double-stranded cDNA molecule or complementary to an mRNA sequence.In specific aspects, antisense nucleic acid molecules are provided thatcomprise a sequence complementary to at least about 10, 25, 50, 100, 250or 500 nucleotides or an entire HPR coding strand, or to only a portionthereof. Nucleic acid molecules encoding fragments, homologs,derivatives and analogs of a HPR protein of SEQ ID NO:2 or antisensenucleic acids complementary to a HPR nucleic acid sequence of SEQ IDNO:1 are additionally provided. Exemplary HPR anti-sense nucleic acidinclude the nucleic acid sequences of SEQ ID NO:3.

In one embodiment, an antisense nucleic acid molecule is antisense to a“coding region” of the coding strand of a nucleotide sequence encodingHPR. The term “coding region” refers to the region of the nucleotidesequence comprising codons which are translated into amino acid residues(e.g., the protein coding region of Arabidopsis thaliana HPR correspondsto SEQ ID NO:2 ). In another embodiment, the antisense nucleic acidmolecule is antisense to a “noncoding region” of the coding strand of anucleotide sequence encoding HPR. The term “noncoding region” refers to5′ and 3′ sequences which flank the coding region that are nottranslated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

Given the coding strand sequences encoding HPR disclosed herein (e.g.SEQ ID NO:1), antisense nucleic acids of the invention can be designedaccording to the rules of Watson and Crick or Hoogsteen base pairing.The antisense nucleic acid molecule can be complementary to the entirecoding region of HPR mRNA, but more preferably is an oligonucleotidethat is antisense to only a portion of the coding or noncoding region ofHPR mRNA. For example, the antisense oligonucleotide can becomplementary to the region surrounding the translation start site ofHPR mRNA. An antisense oligonucleotide can be, for example, about 5, 10,15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis or enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used.

Examples of modified nucleotides that can be used to generate theantisense nucleic acid include: 5-fluorouracil, 5-bromouracil,5-chlorouracil, S-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are generated insitu such that they hybridize with or bind to cellular mRNA and/orgenomic DNA encoding a HPR protein to thereby inhibit expression of theprotein, e.g., by inhibiting transcription and/or translation. Thehybridization can be by conventional nucleotide complementarity to forma stable duplex, or, for example, in the case of an antisense nucleicacid molecule that binds to DNA duplexes, through specific interactionsin the major groove of the double helix. An example of a route ofadministration of antisense nucleic acid molecules of the inventionincludes direct injection at a tissue site. Alternatively, antisensenucleic acid molecules can be modified to target selected cells and thenadministered systemically. For example, for systemic administration,antisense molecules can be modified such that they specifically bind toreceptors or antigens expressed on a selected cell surface, e.g., bylinking the antisense nucleic acid molecules to peptides or antibodiesthat bind to cell surface receptors or antigens. The antisense nucleicacid molecules can also be delivered to cells using the vectorsdescribed herein. To achieve sufficient intracellular concentrations ofantisense molecules, vector constructs in which the antisense nucleicacid molecule is placed under the control of a strong pol I or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an a-anomeric nucleic acid molecule. An ot-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual D-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). Theantisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett215: 327-330).

Such modifications include, by way of nonlimiting example, modifiedbases, and nucleic acids whose sugar phosphate backbones are modified orderivatized. These modifications are carried out at least in part toenhance the chemical stability of the modified nucleic acid, such thatthey may be used, for example, as antisense binding nucleic acids inapplications.

Double Stranded RNA Inhibition (RNAi) by Hairpin Nucleic Acids

Another aspect of the invention pertains to the use of posttranscriptional gene silencing (PTGS) to repress gene expression. Doublestranded RNA can initiate the sequence specific repression of geneexpression in plants and animals. Double stranded RNA is processed toshort duplex oligomers of 21-23 nucleotides in length. These smallinterfering RNA's suppress the expression of endogenous and heterologousgenes in a sequence specific manner (Fire et al. Nature 391:806-811,Carthew, Curr. Opin. in Cell Biol., 13:244-248, Elbashir et al., Nature411:494-498). A RNAi suppressmg construct can be designed in a number ofways, for example, transcription of a inverted repeat which can form along hair pin molecule, inverted repeats separated by a spacer sequencethat could be an unrelated sequence such as GUS or an intron sequence.Transcription of sense and antisense strands by opposing promoters orcotranscription of sense and antisense genes.

HPR Ribozymes and PNA Moieties

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity that are capable of cleaving a single-stranded nucleic acid,such as a mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveHPR mRNA transcripts to thereby inhibit translation of HPR mRNA. Aribozyme having specificity for a HPR-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a HPR DNA disclosedherein (i.e., SEQ ID NO:1). For example, a derivative of a TetrahymenaL-19 IVS RNA can be constructed in which the nucleotide sequence of theactive site is complementary to the nucleotide sequence to be cleaved ina HPR-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; andCech et al. U.S. Pat. No. 5,116,742. Alternatively, HPR mRNA can be usedto select a catalytic RNA having a specific ribonuclease activity from apool of RNA molecules. See, e.g., Bartel et al., (1993) Science261:1411-1418.

Alternatively, HPR gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the HPR(e.g., the HPR promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the HPR gene in target cells.See generally, Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. etal. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays14: 807-15.

In various embodiments, the nucleic acids of HPR can be modified at thebase moiety, sugar moiety or phosphate backbone to improve, e.g., thestability, hybridization, or solubility of the molecule. For example,the deoxyribose phosphate backbone of the nucleic acids can be modifiedto generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg MedChem 4: 5-23). As used herein, the terms “peptide nucleic acids” or“PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which thedeoxyribose phosphate backbone is replaced by a pseudopeptide backboneand only the four natural nucleobases are retained. The neutral backboneof PNAs has been shown to allow for specific hybridization to DNA andRNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup et al. (1996) above; Perry-O'Keefe etal. (1996) PNAS 93: 14670-675.

PNAs of HPR can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs ofHPR can also be used, e.g., in the analysis of single base pairmutations in a gene by, e.g., PNA directed PCR clamping; as artificialrestriction enzymes when used in combination with other enzymes, e.g.,SI nucleases (Hyrup B. (1996) above); or as probes or primers for DNAsequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe(1996), above).

In another embodiment, PNAs of HPR can be modified, e.g., to enhancetheir stability or cellular uptake, by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of HPR can be generated that may combinethe advantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNase H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation ([yrup (1996) above). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996) above and Finn et al. (1996) Nuc Acids Res 24: 3357-63. Forexample, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry, and modified nucleosideanalogs, e.g., 5′-(4-methoxytrityl) amino-5′-deoxy-thymidinephosphoramidite, can be used between the PNA and the 5′ end of DNA (Maget al. (1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupledin a stepwise manner to produce a chimeric molecule with a 5′ PNAsegment and a 3′ DNA segment (Finn et al. (1996) above). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.

HPR Polypeptides

A HPR polypeptide of the invention includes the protein whose sequenceis provided in SEQ ID NO:2. The invention also includes a mutant orvariant protein any of whose residues may be changed from thecorresponding residue shown in SEQ ID NO:2 while still encoding aprotein that maintains its HPR-like activities and physiologicalfunctions, or a functional fragment thereof. In some embodiments, up to20% or more of the residues may be so changed in the mutant or variantprotein. In some embodiments, the HPR polypeptide according to theinvention is a mature polypeptide.

In general, a HPR-like variant that preserves HPR-like function includesany variant in which residues at a particular position in the sequencehave been substituted by other amino acids, and further include thepossibility of inserting an additional residue or residues between tworesidues of the parent protein as well as the possibility of deletingone or more residues from the parent sequence. Any amino acidsubstitution, insertion, or deletion is encompassed by the invention. Infavorable circumstances, the substitution is a conservative substitutionas defined above.

One aspect of the invention pertains to isolated HPR proteins, andbiologically active portions thereof, or derivatives, fragments, analogsor homologs thereof. Also provided are polypeptide fragments suitablefor use as immunogens to raise anti-HPR antibodies. In one embodiment,native HPR proteins can be isolated from cells or tissue sources by anappropriate purification scheme using standard protein purificationtechniques. In another embodiment, HPR proteins are produced byrecombinant DNA techniques. Alternative to recombinant expression, a HPRprotein or polypeptide can be synthesized chemically using standardpeptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which the HPRprotein is derived, or substantially free from chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of HPR protein in whichthe protein is separated from cellular components of the cells fromwhich it is isolated or recombinantly produced. In one embodiment, thelanguage “substantially free of cellular material” includes preparationsof HPR protein having less than about 30% (by dry weight) of non-HPRprotein (also referred to herein as a “contaminating protein”), morepreferably less than about 20% of non-HPR protein, still more preferablyless than about 10% of non-HPR protein, and most preferably less thanabout 5% non-HPR protein. When the HPR protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of HPR protein in which the protein isseparated from chemical precursors or other chemicals that are involvedin the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of HPR protein having less than about 30% (by dry weight)of chemical precursors or non-HPR chemicals, more preferably less thanabout 20% chemical precursors or non-HPR chemicals, still morepreferably less than about 10% chemical precursors or non-HPR chemicals,and most preferably less than about 5% chemical precursors or non-HPRchemicals.

Biologically active portions of a HPR protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the HPR protein, e.g., the amino acidsequence shown in SEQ ID NO:2 that include fewer amino acids than thefull length HPR proteins, and exhibit at least one activity of a HPRprotein, e.g. substrate binding. Typically, biologically active portionscomprise a domain or motif with at least one activity of the HPRprotein. A biologically active portion of a HPR protein can be apolypeptide which is, for example, 10, 25, 50, 100 or more amino acidsin length.

A biologically active portion of a HPR protein of the present inventionmay contain at least one of the above-identified domains conservedbetween the HPR proteins, e.g. Moreover, other biologically activeportions, in which other regions of the protein are deleted, can beprepared by recombinant techniques and evaluated for one or more of thefunctional activities of a native HPR protein.

A biologically active portion or a HPR protein can be the N-terminaldomain of the HPR polypeptide. Alternatively, a biologically activeportion or a HPR protein can be the C-terminal domain of the HPRpolypeptide. Preferably, the biologically active portion comprises atleast 75 amino acids of the C-terminal domain. More preferably, thebiologically active portion comprises at least 25 amino acids of theC-terminal domain. Most preferably, the biologically active portioncomprises at least 10 amino acids of the C-terminal.

In an embodiment, the HPR protein has an amino acid sequence shown inSEQ ID NO:2. In other embodiments, the HPR protein is substantiallyhomologous to SEQ ID NO:2 and retains the functional activity of theprotein of SEQ ID NO:2, yet differs in amino acid sequence due tonatural allelic variation or mutagenesis, as described in detail below.Accordingly, in another embodiment, the HPR protein is a protein thatcomprises an amino acid sequence at least about 45% homologous to theamino acid sequence of S SEQ ID NO:2 and retains the functional activityof the HPR proteins of SEQ ID NO:2.

Determining Homology Between Two or More Sequence

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in either of the sequences being comparedfor optimal alignment between the sequences). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules arehomologous at that position (i.e., as used herein amino acid or nucleicacid “homology” is equivalent to amino acid or nucleic acid “identity”).

The nucleic acid sequence homology may be determined as the degree ofidentity between two sequences. The homology may be determined usingcomputer programs known in the art, such as GAP software provided in theGCG program package. See, Needleman and Wunsch 1970 J Mol Biol 48:443-453. Using GCG GAP software with the following settings for nucleicacid sequence comparison: GAP creation penalty of 5.0 and GAP extensionpenalty of 0.3, the coding region of the analogous nucleic acidsequences referred to above exhibits a degree of identity preferably ofat least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS(encoding) part of the DNA sequence shown in SEQ ID NO:1 .

The term “sequence identity” refers to the degree to which twopolynucleotide or polypeptide sequences are identical on aresidue-by-residue basis over a particular region of comparison. Theterm “percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over that region of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I, in the case of nucleic acids) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the region ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. The term “substantialidentity” as used herein denotes a characteristic of a polynucleotidesequence, wherein the polynucleotide comprises a sequence that has atleast 80 percent sequence identity, preferably at least 85 percentidentity and often 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison region. The term “percentage of positive residues” iscalculated by comparing two optimally aligned sequences over that regionof comparison, determining the number of positions at which theidentical and conservative amino acid substitutions, as defined above,occur in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the region of comparison (i.e., the window size), andmultiplying the result by 100 to yield the percentage of positiveresidues. Chimeric and fusion proteins

The invention also provides HPR chimeric or fusion proteins. As usedherein, a HPR “chimeric protein” or “fusion protein” comprises a HPRpolypeptide operatively linked to a non-HPR polypeptide. An “HPRpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to HPR, whereas a “non-HPR polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinthat is not substantially homologous to the HPR protein, e.g., a proteinthat is different from the HPR protein and that is derived from the sameor a different organism. Within a HPR fusion protein the HPR polypeptidecan correspond to all or a portion of a HPR protein. In one embodiment,a HPR fusion protein comprises at least one biologically active portionof a HPR protein. In another embodiment, a HPR fusion protein comprisesat least two biologically active portions of a HPR protein. Within thefusion protein, the term “operatively linked” is intended to indicatethat the HPR polypeptide and the non-HPR polypeptide are fused in-frameto each other. The non-HPR polypeptide can be fused to the N-terminus orC-terminus of the HPR polypeptide.

A HPR chimeric or fusion protein of the invention can be produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor stagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A HPR-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to theHPR protein.

HPR Antibodies

HPR polypeptides, including chimeric polypeptides, or derivatives,fragments, analogs or homologs thereof, may be utilized as immunogens togenerate antibodies that immunospecifically-bind these peptidecomponents. Such antibodies include, e.g., polyclonal, monoclonal,chimeric, single chain, Fab fragments and a Fab expression library. In aspecific embodiment, fragments of the HPR polypeptides are used asimmunogens for antibody production. Various procedures known within theart may be used for the production of polyclonal or monoclonalantibodies to a HPR polypeptides, or derivative, fragment, analog orhomolog thereof.

For the production of polyclonal antibodies, various host animals may beimmunized by injection with the native peptide, or a synthetic variantthereof, or a derivative of the foregoing. Various adjuvants may be usedto increase the immunological response and include, but are not limitedto, Freund's (complete and incomplete), mineral gels (e.g., aluminumhydroxide), surface active substances (e.g., lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.) andhuman adjuvants such as Bacille Calmette-Guerin and Corynebacteriumparvuni.

For preparation of monoclonal antibodies directed towards a HPRpolypeptides, or derivatives, fragments, analogs or homologs thereof,any technique that provides for the production of antibody molecules bycontinuous cell line culture may be utilized. Such techniques include,but are not limited to, the hybridoma technique (see, Kohler andMilstein, 1975. Nature 256: 495-497); the trioma technique; the humanB-cell hybridoma technique (see, Kozbor, et al., 1983. Immunol Today 4:72) and the EBV hybridoma technique to produce human monoclonalantibodies (see, Cole, et al., 1985. In: Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). Human monoclonalantibodies may be utilized in the practice of the present invention andmay be produced by the use of human hybridomas (see, Cote, et al., 1983.Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cellswith Epstein Barr Virus in vitro (see, Cole, et al., 1985. In MonoclonalAntibodies and Cancer Therapy (Alan R. Liss, Inc., pp. 77-96).

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to a HPR polypeptides (see, e.g.,U.S. Pat. No. 4,946,778). In addition, methodologies can be adapted forthe construction of Fab expression libraries (see, e.g., Huse, et a.,1989. Science 246: 1275-1281) to allow rapid and effectiveidentification of monoclonal Fab fragments with the desired specificityfor a HPR polypeptides or derivatives, fragments, analogs or homologsthereof Antibody fragments that contain the idiotypes to a HPRpolypeptides may be produced by techniques known in the art including,e.g., (i) an F(ab′)₂ fragment produced by pepsin digestion of anantibody molecule; (ii) an Fab fragment generated by reducing thedisulfide bridges of an F(ab′)₂ fragment; (iii) an Fab fragmentgenerated by the treatment of the antibody molecule with papain and areducing agent and (iv) Fv fragments.

In one embodiment, methodologies for the screening of antibodies thatpossess the desired specificity include, but are not limited to,enzyme-linked immunosorbent assay (ELISA) and otherimmunologically-mediated techniques known within the art. In a specificembodiment, selection of antibodies that are specific to a particulardomain of a HPR polypeptides is facilitated by generation of hybridomasthat bind to the fragment of a HPR polypeptides possessing such adomain. Antibodies that are specific for a domain within a HPRpolypeptides, or derivative, fragments, analogs or homologs thereof, arealso provided herein. The anti-HPR polypeptide antibodies may be used inmethods known within the art relating to the localization and/orquantitation of a HPR polypeptide(e.g., for use in measuring levels ofthe peptide within appropriate physiological samples, for use indiagnostic methods, for use in imaging the peptide, and the like).

HPR Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a HPR protein, orderivatives, fragments, analogs or homologs thereof. As used herein, theterm “vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. Exemplary expressionvector constructs include for example the constructs illustrated in FIG.1 and FIG. 2. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication). Other vectors are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors or planttransformation vectors, binary or otherwise, which serve equivalentfunctions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively-linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably-linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the.vector is introduced into the host cell).

The term “regulatory sequence” is intended to includes promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZMOLOGY 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include those thatdirect constitutive expression of a nucleotide sequence in many types ofhost cell and those that direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences).Examples of suitable promoters include for example constitutivepromoters, ABA inducible promoters, tissue specific promters or guardcell specific promoters. It will be appreciated by those skilled in theart that the design of the expression vector can depend on such factorsas the choice of the host cell to be transformed, the level ofexpression of protein desired, etc. The expression vectors of theinvention can be introduced into host cells to thereby produce proteinsor peptides, including fusion proteins or peptides, encoded by nucleicacids as described herein (e.g., HPR proteins, mutant forms of HPRproteins, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of HPR proteins in prokaryotic or eukaryotic cells. Forexample, HPR proteins can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors)yeast cells, plant cells or mammalian cells. Suitable host cells arediscussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out inEscherichia coli with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionproteins. Fusion vectors add a number of amino acids to a proteinencoded therein, usually to the amino terminus of the recombinantprotein. Such fusion vectors typically serve three purposes: (i) toincrease expression of recombinant protein; (ii) to increase thesolubility of the recombinant protein; and (iii) to aid in thepurification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterolinase. Typical fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et.al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. See, e.g., Gottesman,GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,San Diego, Calif (1990) 119-128. Another strategy is to alter thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (see, e.g., Wada, et al., 1992.Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acidsequences of the invention can be carried out by standard DNA synthesistechniques.

In another embodiment, the HPR expression vector is a yeast expressionvector. Examples of vectors for expression in yeast Saccharomycescerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234),pMFa (uijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz etal., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

Alternatively, HPR can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors available for expression ofproteins in cultured insect cells (e.g., SF9 cells) include the pAcseries (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVLseries (Lucklow and Summers, 1989. Virology 170: 31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840)and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, adenovirus 2, cytomegalovirus, andsimian virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 ofSambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In yet another embodiment, a nucleic acid of the invention is expressedin plants cells using a plant expression vector. Examples of plantexpression vectors systems include tumor inducing (Ti) plasmid orportion thereof found in Agrobacterium, cauliflower mosaic virus (CAMV)DNA and vectors such as pBI121 .

For expression of HPR in plants, the recombinant expression cassettewill contain in addition to the HPR nucleic acids, a plant promoterregion, a transcription initiation site (if the coding sequence totranscribed lacks one), and a transcription termination/polyadenylationsequence. The termination/polyadenylation region may be obtained fromthe same gene as the promoter sequence or may be obtained from differentgenes. Unique restriction enzyme sites at the 5′ and 3′ ends of thecassette are typically included to allow for easy insertion into apre-existing vector.

Examples of suitable promotors include promoters from plant viruses suchas the 35S promoter from cauliflower mosaic virus (CaMV). Odell, et al.,Nature, 313: 810-812 (1985). and promoters from genes such as rice actin(McElroy, et al., Plant Cell, 163-171 (1990)); ubiquitin (Christensen,et al., Plant Mol. Biol., 12: 619-632 (1992); and Christensen, et al.,Plant Mol. Biol., 18: 675-689 (1992)); pEMU (Last, et al., Theor. Appl.Genet., 81: 581-588 (1991)); MAS (Velten, et al., EMBO J., 3: 2723-2730(1984)); maize H3 histone (Lepetit, et al., Mol. Gen. Genet., 231:276-285 (1992); and Atanassvoa, et al., Plant Journal, 2(3): 291-300(1992)), the 5′- or 3′-promoter derived from T-DNA of Agrobacteriumtumefaciens, the Smas promoter, the cinnamyl alcohol dehydrogenasepromoter (U.S. Pat. No. 5,683,439), the Nos promoter, the rubiscopromoter, the GRP1-8 promoter, ALS promoter, (WO 96/30530), a syntheticpromoter, such as, Rsyn7, SCP and UCP promoters,ribulose-1,3-diphosphate carboxylase, fruit-specific promoters, heatshock promoters, seed-specific promoters and other transcriptioninitiation regions from various plant genes, for example, include thevarious opine initiation regions, such as for example, octopine,mannopine, and nopaline.

Additional regulatory elements that may be connected to a HPR encodingnucleic acid sequence for expression in plant cells include terminators,polyadenylation sequences, and nucleic acid sequences encoding signalpeptides that permit localization within a plant cell or secretion ofthe protein from the cell. Such regulatory elements and methods foradding or exchanging these elements with the regulatory elements HPRgene are known, and include, but are not limited to, 3′ terminationand/or polyadenylation regions such as those of the Agrobacteriumtumefaciens nopaline synthase (nos) gene (Bevan, et al., Nucl. AcidsRes., 12: 369-385 (1983)); the potato proteinase inhibitor II (PINII)gene (Keil, et al., Nucl. Acids Res., 14: 5641-5650 (1986) and herebyincorporated by reference); and An, et al., Plant Cell, 1: 115-122(1989)); and the CaMV 19S gene (Mogen, et al., Plant Cell, 2: 1261-1272(1990)).

Plant signal sequences, including, but not limited to, signal-peptideencoding DNA/RNA sequences which target proteins to the extracellularmatrix of the plant cell (Dratewka-Kos, et al., J. Biol. Chem., 264:4896-4900 (1989)) and the Nicotiana plumbaginifolia extension gene(DeLoose, et al., Gene, 99: 95-100 (1991)), or signal peptides whichtarget proteins to the vacuole like the sweet potato sporamin gene(Matsuka, et al., Proc. Nat'l Acad. Sci. (ISA), 88: 834 (1991)) and thebarley lectin gene (Wilkins, et al., Plant Cell, 2: 301-313 (1990)), orsignals which cause proteins to be secreted such as that of PRIb (Lind,et al., Plant Mol. Biol., 18: 47-53 (1992)), or those which targetproteins to the plastids such as that of rapeseed enoyl-ACP reductase(Verwaert, et al., Plant Mol. Biol., 26: 189-202 (1994)) are useful inthe invention.

In another embodiment, the recombinant expression vector is capable ofdirecting expression of the nucleic acid preferentially in a particularcell type (e.g., tissue-specific regulatory elements are used to expressthe nucleic acid). Tissue-specific regulatory elements are known in theart. Especially useful in connection with the nucleic acids of thepresent invention are expression systems which are operable in plants.These include systems which are under control of a tissue-specificpromoter, as well as those which involve promoters that are operable inall plant tissues.

Organ-specific promoters are also well known. For example, the patatinclass I promoter is transcriptionally activated only in the potato tuberand can be used to target gene expression in the tuber (Bevan, M., 1986,Nucleic Acids Research 14:4625-4636). Another potato-specific promoteris the granule-bound starch synthase (GBSS) promoter (Visser, R. G. R,et al., 1991, Plant Molecular Biology 17:691-699).

Other organ-specific promoters appropriate for a desired target organcan be isolated using known procedures. These control sequences aregenerally associated with genes uniquely expressed in the desired organ.In a typical higher plant, each organ has thousands of mRNAs that areabsent from other organ systems (reviewed in Goldberg, P., 1986, Trans.R. Soc. London B314:343).

For in situ production of the antisense mRNA of HPR, those regions ofthe HPR gene which are transcribed into HPR mRNA, including theuntranslated regions thereof, are inserted into the expression vectorunder control of the promoter system in a reverse orientation. Theresulting transcribed mRNA is then complementary to that normallyproduced by the plant.

The resulting expression system or cassette is ligated into or otherwiseconstructed to be included in a recombinant vector which is appropriatefor plant transformation. The vector may also contain a selectablemarker gene by which transformed plant cells can be identified inculture. Usually, the marker gene will encode antibiotic resistance.These markers include resistance to G418, hygromycin, bleomycin,kanamycin, and gentamicin. After transforming the plant cells, thosecells having the vector will be identified by their ability to grow on amedium containing the particular antibiotic. Replication sequences, ofbacterial or viral origin, are generally also included to allow thevector to be cloned in a bacterial or phage host, preferably a broadhost range prokaryotic origin of replication is included. A selectablemarker for bacteria should also be included to allow selection ofbacterial cells bearing the desired construct. Suitable prokaryoticselectable markers also include resistance to antibiotics such askanamycin or tetracycline.

Other DNA sequences encoding additional functions may also be present inthe vector, as is known in the art. For instance, in the case ofAgrobacterium transformations, T-DNA sequences will also be included forsubsequent transfer to plant chromosomes.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a polypeptide ofthe invention encoded in a an open reading frame of a polynucleotide ofthe invention. Accordingly, the invention further provides methods forproducing a polypeptide using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of invention(into which a recombinant expression vector encoding a polypeptide ofthe invention has been introduced) in a suitable medium such that thepolypeptide is produced. In another embodiment, the method furthercomprises isolating the polypeptide from the medium or the host cell.

A number of types of cells may act as suitable host cells for expressionof a polypeptide encoded by an open reading frame in a polynucleotide ofthe invention. Plant host cells include, for example, plant cells thatcould function as suitable hosts for the expression of a polynucleotideof the invention include epidermal cells, mesophyll and other groundtissues, and vascular tissues in leaves, stems, floral organs, and rootsfrom a variety of plant species, such as Arabidopsis thaliana, Nicotianatabacum, Brassica napus, Zea mays, Oryza sativa, Gossypium hirsutum andGlycine max.

Alternatively, it may be possible to produce a polypeptide in lowereukaryotes such as yeast or in prokaryotes such as bacteria. Potentiallysuitable yeast strains include Saccharomiyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous proteins. Potentially suitablebacterial strains include Eschlerichia coli, Bacillus subtilis,Salmonella typhiniururn, or any bacterial strain capable of expressingheterologous polypeptides. If the polypeptide is made in yeast orbacteria, it may be necessary to modify the polypeptide producedtherein, for example by phosphorylation or glycosylation of theappropriate sites, in order to obtain a functional polypeptide, if thepolypeptide is of sufficient length and conformation to have activity.Such covalent attachments may be accomplished using known chemical orenzymatic methods.

A polypeptide may be prepared by culturing transformed host cells underculture conditions suitable to express the recombinant protein. Theresulting expressed polypeptide or protein may then be purified fromsuch culture (e.g., from culture medium or cell extracts) using knownpurification processes, such as gel filtration and ion exchangechromatography. The purification of the polypeptide or protein may alsoinclude an affinity column containing agents which will bind to theprotein; one or more column steps over such affinity resins asconcanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GASepharose®; one or more steps involving hydrophobic interactionchromatography using such resins as phenyl ether, butyl ether, or propylether; or immunoaffinity chromatography.

Alternatively, a polypeptide or protein may also be expressed in a formwhich will facilitate purification. For example, it may be expressed asa fusion protein containing a six-residue histidine tag. Thehistidine-tagged protein will then bind to a Ni-affinity column. Afterelution of all other proteins, the histidine-tagged protein can beeluted to achieve rapid and efficient purification. One or morereverse-phase high performance liquid chromatography (RP-HPLC) stepsemploying hydrophobic RP-HPLC media, e.g., silica gel having pendantmethyl or other aliphatic groups, can be employed to further purify apolypeptide. Some or all of the foregoing purification steps, in variouscombinations, can also be employed to provide a substantiallyhomogeneous isolated recombinant polypeptide. The protein or polypeptidethus purified is substantially free of other plant proteins orpolypeptides and is defined in accordance with the present invention as“isolated.”

Transformed Plants Cells and Transgenic Plants

The invention includes protoplast, plants cells, plant tissue and plants(e.g., monocots and dicots transformed with a HPR promoter-geneconstruct, a vector (e.g., expression vector) containing a HPRpromoter-gene construct, a HPR nucleic acid (i.e, sense or antisense), avector containing a HPR nucleic acid (i.e, sense or antisense)or anexpression vector containing a HPR nucleic acid (i.e, sense orantisense). As used herein, “plant” is meant to include not only a wholeplant but also a portion thereof (ie., cells, and tissues, including forexample, leaves, stems, shoots, roots, flowers, fruits and seeds).

The plant can be any plant type including, for example, species from thegenera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago,Onobrychis, Trifolium, Trigonella, Vigiza, Citrus, Linumn, Geranium,Mainihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa,Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia,Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Peynisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum,Sorghum, Gossypium, Picea, Caco, and Populus.

In some aspects of the invention, the transformed plant is resistant tobiotic and abiotic stresses, e.g., chilling stress, heat stress, saltstress, water stress (e.g., drought), photo-oxidative stress, disease,grazing pests and wound healing. Additionally, the invention alsoincludes a transgenic plant that is resistant to pathogens such as forexample fungi, bacteria, nematodes, viruses and parasitic weeds.Alternatively, the transgenic plant is resistant to herbicides or hasaltered senescence. The transgenic plant has an increase in yield,productivity, biomass or ABA sensitivity. By resistant is meant theplant grows under stress conditions (e.g., high salt, decreased water,low temperatures) or under conditions that normally inhibit, to somedegree, the growth of an untransformed plant. Methodologies to determineplant growth or response to stress include for example, heightmeasurements, weight measurements, leaf area, ability to flower, wateruse, transpiration rates and yield.

The invention also includes cells, tissues, including for example,leaves, stems, shoots, roots, flowers, fruits and seeds and the progenyderived from the tranformed plant.

Numerous methods for introducing foreign genes into plants are known andcan be used to insert a gene into a plant host, including biological andphysical plant transformation protocols. See, for example, Miki et al.,(1993) “Procedure for Introducing Foreign DNA into Plants”, In: Methodsin Plant Molecular Biology and Biotechnology, Glick and Thompson, eds.,CRC Press, Inc., Boca Raton, pages 67-88 and Andrew Bent in, Clough S Jand Bent A F, 1998. Floral dipping: a simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana. Themethods chosen vary with the host plant, and include chemicaltransfection methods such as calcium phosphate, polyethylene glycol(PEG) transformation, microorganism-mediated gene transfer such asAgrobacterium (Horsch, et al., Science, 227: 1229-31 (1985)),electroporation, protoplast transformation, micro-injection, flowerdipping and biolistic bombardment.

Agrobacterium-Mediated Transformation

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectfully, carry genesresponsible for genetic transformation of plants. See, for example,Kado, Crit. Rev. Plant Sci., 10: 1-32 (1991). Descriptions of theAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided in Gruber et al., supra; and Moloney, et al, PlantCell Reports, 8: 238-242 (1989).

Transgenic Arabidopsis plants can be produced easily by the method ofdipping flowering plants into an Agrobacterium culture, based on themethod of Andrew Bent in, Clough S J and Bent A F, 1998. Floral dipping:a simplified method for Agrobacterium-mediated transformation ofArabidopsis thaliana. Wild type plants are grown until the: plant hasboth developing flowers and open flowers. The plant are inverted for 1minute into a solution of Agrobacterium culture carrying the appropriategene construct. Plants are then left horizontal in a tray and keptcovered for two days to maintain humidity and then righted and bagged tocontinue growth and seed development. Mature seed is bulk harvested.

Direct Gene Transfer

A generally applicable method of plant transformation ismicroprojectile-mediated transformation, where DNA is carried on thesurface of microprojectiles measuring about 1 to 4 mu.m. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate the plant cell walls and membranes. (Sanford, etal., Part. Sci. Technol., 5: 27-37 (1987); Sanford, Trends Biotech, 6:299-302 (1988); Sanford, Physiol. Plant, 79: 206-209 (1990); Klein, etal., Biotechnology, 10: 286-291 (1992)).

Another method for physical delivery of DNA to plants is sonication oftarget cells as described in Zang, et al., BioTechnology, 9: 996-996(1991). An alternative transformation method utilizing sonication isdescribed in U.S. Pat. No. 5,693,512 as described by Finer, J. J. andTrick, H. N. Alternatively, liposome or spheroplast fusions have beenused to introduce expression vectors into plants. See, for example,Deshayes, et al., EMBO J., 4: 2731-2737 (1985); and Christou, et al.,Proc. Nat'l. Acad. Sci. (USA), 84: 3962-3966 (1987). Direct uptake ofDNA into protoplasts using CaCl.sub.2 precipitation, polyvinyl alcoholor poly-L-ornithine have also been reported. See, for example, Hain, etal., Mol. Gen. Genet., 199: 161 (1985); and Draper, et al., Plant CellPhysiol., 23: 451-458 (1982).

Electroporation of protoplasts and whole cells and tissues has also beendescribed. See, for example, Donn, et al., (1990) In: Abstracts of theVIIth Int;l. Congress on Plant Cell and Tissue Culture IAPTC, A2-38,page 53; D'Halluin et al., Plant Cell, 4: 1495-1505 (1992); and Spenceret al., Plant Mol. Biol., 24: 51-61 (1994).

Alternatively one can use a non-particle biolistic bombardmenttransformation method. An example of non-particle biolistictransformation is given in U.S. Patent Application 20010026941. Thismethod has been used to produce transgenic Glycine max and Zea maizeplants. Viable plants are propagated and homozygous lines are generated.

Particle Wounding/Agrobacterium Delivery

Another useful basic transformation protocol involves a combination ofwounding by particle bombardment, followed by use of Agrobacterium forDNA delivery, as described by Bidney, et al., Plant Mol. Biol., 18:301-31 (1992). Useful plasmids for plant transformation include Bin 19.See Bevan, Nucleic Acids Research, 12: 8711-8721 (1984), and herebyincorporated by reference.

In general, the intact meristem transformation method involves imbibingseed for 24 hours in the dark, removing the cotyledons and root radical,followed by culturing of the meristem explants. Twenty-four hours later,the primary leaves are removed to expose the apical meristem. Theexplants are placed apical dome side up and bombarded, e.g., twice withparticles, followed by co-cultivation with Agrobacterium. To start theco-cultivation for intact meristems, Agrobacterium is placed on themeristem. After about a 3-day co-cultivation period the meristems aretransferred to culture medium with cefotaxime plus kanamycin for theNPTII selection.

The split meristem method involves imbibing seed, breaking of thecotyledons to produce a clean fracture at the plane of the embryonicaxis, excising the root tip and then bisecting the explantslongitudinally between the primordial leaves. The two halves are placedcut surface up on the medium then bombarded twice with particles,followed by co-cultivation with Agrobacterium. For split meristems,after bombardment, the meristems are placed in an Agrobacteriumsuspension for 30 minutes. They are then removed from the suspensiononto solid culture medium for three day co-cultivation. After thisperiod, the meristems are transferred to fresh medium with cefotaximeplus kanamycin for selection.

Transfer by Plant Breeding

Alternatively, once a single transformed plant has been obtained by theforegoing recombinant DNA method, conventional plant breeding methodscan be used to transfer the gene and associated regulatory sequences viacrossing and backcrossing. Such intermediate methods will comprise thefurther steps of: (1) sexually crossing the transgenic plant with aplant from a second taxon; (2) recovering reproductive material from theprogeny of the cross; and (3) growing transgenic plants from thereproductive material. Where desirable or necessary, the agronomiccharacteristics of the second taxon can be substantially preserved byexpanding this method to include the further steps of repetitively: (1)backcrossing the transgenic progeny with non-transgenic plants from thesecond taxon; and (2) selecting for expression of an associated markergene among the progeny of the backcross, until the desired percentage ofthe characteristics of the second taxon are present in the progeny alongwith the gene or genes imparting marker gene trait.

By the term “taxon” herein is meant a unit of botanical classification.It thus includes, genus, species, cultivars, varieties, variants andother minor taxonomic groups which lack a consistent nomenclature.

Regeneration of Transformants

The development or regeneration of plants from either single plantprotoplasts or various explants is well known in the art (Weissbach andWeissbach, 1988). This regeneration and growth process typicallyincludes the steps of selection of transformed cells, culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983). In particular, U.S. Pat.No. 5,349,124 (specification incorporated herein by reference) detailsthe creation of genetically transformed lettuce cells and plantsresulting therefrom which express hybrid crystal proteins conferringinsecticidal activity against Lepidopteran larvae to such plants.

This procedure typically produces shoots within two to four months andthose shoots are then transferred to an appropriate root-inducing mediumcontaining the selective agent and an antibiotic to prevent bacterialgrowth. Shoots that rooted in the presence of the selective agent toform plantlets are then transplanted to soil or other media to allow theproduction of roots. These procedures vary depending upon the particularplant strain employed, such variations being well known in the art.

Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, or pollen obtained from the regeneratedplants is crossed to seed-grown plants of agronomically important,preferably inbred lines. Conversely, pollen from plants of thoseimportant lines is used to pollinate regenerated plants. A transgenicplant of the present invention containing a desired polypeptide iscultivated using methods well known to one skilled in the art.

A preferred transgenic plant is an independent segregant and cantransmit the HPR gene and its activity to its progeny. A more preferredtransgenic plant is homozygous for the gene, and transmits that gene toall of its offspring on sexual mating. Seed from a transgenic plant maybe grown in the field or greenhouse, and resulting sexually maturetransgenic plants are self-pollinated to generate true breeding plants.The progeny from these plants become true breeding lines that areevaluated for increased expression of the HPR transgene.

Method of Producing Transgenic Plants

Also included in the invention are methods of producing a transgenicplant.

A transgenic plant is produced by introducing into one or more plantcells a HPR promoter-gene construct according to the invention (asdescribed above) to generate a transgenic cell and regenerating atransgenic plant from the transgenic cell. The transgenic plant has analtered level of expression of a protein of interest. For example,expression of the protein level is at an increased level compared to anormal control level. Alternatively, the transgenic plant expresses aprotein of interest at a decreased level compared a normal controllevel. By normal control level is meant a level of gene/proteinexpression detected in a normal, untransformed plant (i.e., wild-type).

For example, when a plant cell is transformed with a HPR-gene constructcontaining a non-translatable mRNA molecule of a gene encoding a proteinof interest, the resulting transgenic plant has a decreased level ofexpression compared to a wild-type plant. Similarly, when a plant cellis transformed with a HPR-gene construct containing a nucleic acidencoding for a gene of interest, the resulting transgenic plant has anincrease level of expression compared to a wild-type plant.

Expression of the protein of interest is detected and measured at thenucleic acid level using techniques well known to one of ordinary skillin the art and include amplification-based detection methods such asreverse-transcription based polymerase chain reaction. Expression of heprotein of interest is also determined at the protein level, i.e., bymeasuring the levels of polypeptides encoded by the gene products. Suchmethods are well known in the art and include, e.g., immunoassays basedon antibodies to proteins encoded by the genes.

Transgenic plants are also produced by introducing into one or moreplant cells a compound that alters hydroxypyruvate reductase expressionor activity in the plant to generate a transgenic plant cell andregenerating a transgenic plant from the transgenic cell. In someaspects the compound increases alters hydroxypyruvate reductaseexpression or activity. Alternatively, the compound decrease altershydroxypyruvate reductase expression or activity. The compound can be,e.g., (i) a hydroxypyruvate reductase polypeptide; (ii) a nucleic acidencoding a hydroxypyruvate reductase polypeptide; (iii) a nucleic acidthat increases expression of a nucleic acid that encodes ahydroxypyruvate reductase polypeptide ; (iv) a nucleic acid thatdecreases the expression of a nucleic acid that encodes ahydroxypyruvate reductase polypeptide; (v) a hydroxypyruvate reductaseantisense nucleic acid and derivatives, fragments, analogs and homologsthereof A nucleic acid that increases expression of a nucleic acid thatencodes a hydroxypyruvate reductase polypeptide includes, e.g.,promoters, enhancers. The nucleic acid can be either endogenous orexogenous. Preferably, the compound is a hydroxypyruvate reductasepolypeptide or a nucleic acid encoding a hydroxypyruvate reductasepolypeptide. For example the compound comprises the nucleic acidsequence of SEQ ID NO:1 or a fragment thereof. Alternatively, thecompound is a hydroxypyruvate reductase antisense nucleic acid. Forexample the compound comprises the nucleic acid sequence of SEQ ID NO:3.

In various aspects the transgenic plant produced by the methods of theinvention has an altered phenotype as compared to a wild type plant(i.e., untransformed). By altered phenotype is meant that the plant hasa one or more characteristic that is different from the wild type plant.For example, the transgenic plant has an increased resistence to stress.Increased stress resistance is meant that the transgenic plant can growunder stress conditions (e.g., high salt, decreased water, lowtemperatures, high temperatures) or under conditions that normallyinhibit the growth of an untransformed Stresses include, for example,chilling stress, heat stress, heat shock, salt stress, water stress(i.e, drought), photo-oxidative stress, nutritional stress, disease,grazing pests, wound healing, pathogens such as for example fungi,bacteria, nematodes, viruses or parasitic weed and herbicides.Methodologies to determine plant growth or response to stress includefor example, height measurements, weight or biomass measurements, leafarea or number, ability to flower, water use, transpiration rates andyield. Alternatively, the transformed plant has an increased (i.e.,enhanced) ABA sensitivity. The enhanced ABA sensitivity is at theseedling growth stage. Alternatively, the enhanced ABA sensitivity is atthe mature plant stage. Additional altered phenotypes include forexample, enhanced vegetative growth (e.g., increased leaf number,thickness and overall biomass), delayed reproductive growth (e.g.,flowering later); enhanced seedling vigor (e.g., increased root biomassand length), enhanced lateral root formation and therefore soilpenetration more extensive vascular system resulting in an enhancedtransport system.

The plant cell from a dicot or a monocot plant. The plant is any planttype including, for example, species from the genera Cucurbita, Rosa,Vitis, Juglans, Fragaria, Lotus, Medicago, Otiobrychis, Tnifolium,Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus,Arabidopsis, Brassica, Raphaizus, Sinapis, Atropa, Capsicum, Datura,Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis,Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Paniieum, Pennisetum,Raizunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum,Sorghum, Gossypium, Picea, Caco, and Populus.

Screening Methods

The isolated nucleic acid molecules of the invention can be used toexpress HPR protein (e.g., via a recombinant expression vector in a hostcell), to detect HPR mRNA (e.g., in a biological sample) or a geneticlesion in a HPR gene, and to modulate HPR activity, as describedfurther, below. In addition, the HPR proteins can be used to screencompounds that modulate the HPR protein activity or expression. Inaddition, the anti-HPR antibodies of the invention can be used to detectand isolate HPR proteins and modulate HPR activity.

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)that bind to HPR proteins or have a stimulatory or inhibitory effect on,e.g., HPR protein expression or HPR protein activity. The invention alsoincludes compounds identified in the screening assays described herein.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to a HPR protein or polypeptide orbiologically-active portion thereof. The test compounds of the inventioncan be obtained using any of the numerous approaches in combinatoriallibrary methods known in the art, including: biological libraries;spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the “one-beadone-compound” library method; and synthetic library methods usingaffinity chromatography selection. The biological library approach islimited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds. See, e.g., Lam, 1997. Anticancer Drug Designi 12: 145.

A “small molecule” as used herein, is meant to refer to a compositionthat has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be, e.g., nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic or inorganic molecules. Libraries of chemical and/or biologicalmixtures, such as fungal, bacterial, or algal extracts, are known in theart and can be screened with any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt, et al., 1993. Proc. Natl.Acad. Sci. US.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci.U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho,et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed.Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (e.g., Houghten,1992. Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354:82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner, U.S. Pat. No. 5,233,409),plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89: 1865-1869)or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990.Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci.U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner,U.S. Pat. No. 5,233,409.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a HPR protein, or a biologically-active portion thereof, iscontacted with a test compound and the ability of the test compound tobind to a HPR protein determined. The cell, for example, can be ofmammalian origin, plant cell or a yeast cell. Determining the ability ofthe test compound to bind to the HPR protein can be accomplished, forexample, by coupling the test compound with a radioisotope or enzymaticlabel such that binding of the test compound to the HPR protein orbiologically-active portion thereof can be determined by detecting thelabeled compound in a complex. For example, test compounds can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, test compounds can beenzymatically-labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product. Inone embodiment, the assay comprises contacting a cell which expresses aHPR protein, or a biologically-active portion thereof, with a knowncompound which binds HPR to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with a HPR protein, wherein determining the abilityof the test compound to interact with a HPR protein comprisesdetermining the ability of the test compound to preferentially bind toHPR protein or a biologically-active portion thereof as compared to theknown compound.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a HPR protein, or a biologically-activeportion thereof, with a test compound and determining the ability of thetest compound to modulate (e.g. stimulate or inhibit) the activity ofthe HPR protein or biologically-active portion thereof. Determining theability of the test compound to modulate the activity of HPR or abiologically-active portion thereof can be accomplished, for example, bydetermining the ability of the HPR protein to bind to or interact with aHPR target molecule. As used herein, a “target molecule” is a moleculewith which a HPR protein binds or interacts in nature, for example, amolecule on the surface of a cell which expresses a HPR interactingprotein, a molecule on the surface of a second cell, a molecule in theextracellular milieu, a molecule associated with the internal surface ofa cell membrane or a cytoplasmic molecule. A HPR target molecule can bea non-HPR molecule or a HPR protein or polypeptide of the invention Inone embodiment, a HPR target molecule is a component of a signaltransduction pathway that facilitates transduction of an extracellularsignal (e.g. a signal generated by binding of a compound to amembrane-bound molecule) through the cell membrane and into the cell.The target, for example, can be a second intercellular protein that hascatalytic activity or a protein that facilitates the association ofdownstream signaling molecules with HPR.

Determining the ability of the HPR protein to bind to or interact with aHPR target molecule can be accomplished by one of the methods describedabove for determining direct binding. In one embodiment, determining theability of the HPR protein to bind to or interact with a HPR targetmolecule can be accomplished by determining the activity of the targetmolecule. For example, the activity of the target molecule can bedetermined by detecting induction of a cellular second messenger of thetarget (i.e. intracellular Ca²⁺, diacylglycerol, IP₃, etc.), detectingcatalytic/enzymatic activity of the target an appropriate substrate,detecting the induction of a reporter gene (comprising a HPR-responsiveregulatory element operatively linked to a nucleic acid encoding adetectable marker, e.g., luciferase), or detecting a cellular response,for example, cell survival, cellular differentiation, or cellproliferation.

In yet another embodiment, an assay of the invention is a cell-freeassay comprising contacting a HPR protein or biologically-active portionthereof with a test compound and determining the ability of the testcompound to bind to the HPR protein or biologically-active portionthereof. Binding of the test compound to the HPR protein can bedetermined either directly or indirectly as described above. In one suchembodiment, the assay comprises contacting the HPR protein orbiologically-active portion thereof with a known compound which bindsHPR to form an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith a HPR protein, wherein determining the ability of the test compoundto interact with a HPR protein comprises determining the ability of thetest compound to preferentially bind to HPR or biologically-activeportion thereof as compared to the known compound.

In still another embodiment, an assay is a cell-free assay comprisingcontacting HPR protein or biologically-active portion thereof with atest compound and determining the ability of the test compound tomodulate (e.g. stimulate or inhibit) the activity of the HPR protein orbiologically-active portion thereof. Determining the ability of the testcompound to modulate the activity of HPR can be accomplished, forexample, by determining the ability of the HPR protein to bind to a HPRtarget molecule by one of the methods described above for determiningdirect binding. In an alternative embodiment, determining the ability ofthe test compound to modulate the activity of HPR protein can beaccomplished by determining the ability of the HPR protein furthermodulate a HPR target molecule. For example, the catalytic/enzymaticactivity of the target molecule on an appropriate substrate can bedetermined as described above.

In yet another embodiment, the cell-free assay comprises contacting theHPR protein or biologically-active portion thereof with a known compoundwhich binds HPR protein to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with a HPR protein, wherein determining the abilityof the test compound to interact with a HPR protein comprisesdetermining the ability of the HPR protein to preferentially bind to ormodulate the activity of a HPR target molecule.

The cell-free assays of the invention are amenable to use of both thesoluble form or the membrane-bound form of HPR protein. In the case ofcell-free assays comprising the membrane-bound form of HPR protein, itmay be desirable to utilize a solubilizing agent such that themembrane-bound form of HPR protein is maintained in solution. Examplesof such solubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate(CHAPSO).

In more than one embodiment of the above assay methods of the invention,it may be desirable to immobilize either HPR protein or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to HPR protein, or interaction of HPRprotein with a target molecule in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided that adds a domain that allows one orboth of the proteins to be bound to a matrix. For example, GST-HPRfusion proteins or GST-target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, that are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or HPR protein, and the mixture is incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and p”). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described, supra. Alternatively,the complexes can be dissociated from the matrix, and the level of HPRprotein binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either the HPRprotein or its target molecule can be immobilized utilizing conjugationof biotin and streptavidin. Biotinylated HPR protein or target moleculescan be prepared from biotin-NHS (N-hydroxy-succinimide) using techniqueswell-known within the art (e.g. biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Alternatively, antibodies reactive withHPR protein or target molecules, but which do not interfere with bindingof the HPR protein to its target molecule, can be derivatized to thewells of the plate, and unbound target or HPR protein trapped in thewells by antibody conjugation. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theHPR protein or target molecule, as well as enzyme-linked assays thatrely on detecting an enzymatic activity associated with the HPR proteinor target molecule.

In another embodiment, modulators of HPR protein expression areidentified in a method wherein a cell is contacted with a candidatecompound and the expression of HPR mRNA or protein in the cell isdetermined. The level of expression of HPR mRNA or protein in thepresence of the candidate compound is compared to the level ofexpression of HPR mRNA or protein in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof HPR mRNA or protein expression based upon this comparison. Forexample, when expression of HPR mRNA or protein is greater (i.e.,statistically significantly greater) in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of HPR mRNA or protein expression. Alternatively, whenexpression of HPR mRNA or protein is less (statistically significantlyless) in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of HPR mRNA or proteinexpression. The level of HPR mRNA or protein expression in the cells canbe determined by methods described herein for detecting HPR mRNA orprotein.

In yet another aspect of the invention, the HPR proteins can be used as“bait proteins” in a two-hybrid assay or three hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos, et al., 1993. Cell 72: 223-232; Madura,et al., 1993. J Biol. Chem. 268: 12046-12054; Bartel, et al., 1993.Biotechniques 14: 920-924; Iwabuchi, et al., 1993. Oncogene 8:1693-1696; and Brent WO 94/10300), to identify other proteins that bindto or interact with HPR (“HPR-binding proteins” or “HPR-bp”) andmodulate HPR activity. Such HPR-binding proteins are also likely to beinvolved in the propagation of signals by the HPR proteins as, forexample, upstream or downstream elements of the HPR pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for HPR is fused to agene encoding the DNA binding domain of a known transcription factor(e.g., GAL-4). In the other construct, a DNA sequence, from a library ofDNA sequences, that encodes an unidentified protein (“prey” or “sample”)is fused to a gene that codes for the activation domain of the knowntranscription factor. If the “bait” and the “prey” proteins are able tointeract, in vivo, forming a HPR-dependent complex, the DNA-binding andactivation domains of the transcription factor are brought into closeproximity. This proximity allows transcription of a reporter gene (e.g.,LacZ) that is operably linked to a transcriptional regulatory siteresponsive to the transcription factor. Expression of the reporter genecan be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genethat encodes the protein which interacts with HPR.

In yet another aspect of the invention are methods which utilize thetransgenic plants of the invention to identify HPR-interactingcomponents via genetic screening protocols. These components can be forexample, regulatory elements which modify HPR-gene expression,interacting proteins which directly modify HPR activity or interactingproteins which modify components of the same signal transduction pathwayand thereby exert an effect on the expression or activity of HPR.Briefly, genetic screening protocols are applied to the transgenicplants of the invention and in so doing identify related genes which arenot identified using a wild type background for the screen. For examplean activation tagged library (Weigel, et al., 2000. Plant Physiol. 122:1003-1013), can be produced using the transgenic plants of the inventionas the genetic background. Plants are then screened for alteredphenotypes from that displayed by the parent plants. Alternative methodsof generating libraries from the transgenic plants of the invention canbe used, for example, chemical or irradiation induced mutations,insertional inactivation or insertional activation methods.

The invention further pertains to novel agents identified by theaforementioned screening assays and uses thereof.

EXAMPLE 1 Identification of line 28.1

Lines of Arabidopsis thaliana transgenic for the pRD29A-anti-FTAconstruct were screened for a drought tolerant phenotype. The phenotypedisplayed by this particular line was that of a drought susceptibleplant. Additionally, the line was found to display ABA insensitivecharacteristics. The line 28.1 could represent an insertional mutant ina related gene of the ABA pathway and or the drought tolerance pathway.

EXAMPLE 2 Identification of HPR Gene

The site of the T-DNA insertion was identified using a PCR approach thatamplified from a known site within the T-DNA to an annealed linker. Alibrary of genomic DNA fragments was produced and screened as follows.Genomic DNA was isolated from Line 28.1 and digested by the restrictionenzyme EcoRV. Two oligonucleotide adapters identified by SEQ ID NO:17and SEQ ID NO:18 were mixed to a final concentration of 25 μM each. Theywere annealed to form a DNA linker by incubating at 95° C. for 1 min andcooling at room temperature. The linker was then ligated to the EcoRVdigested genomic DNA.

A first PCR reaction utilized primers identified by SEQ ED NO:19 and SEQID NO:20. The sequence of SEQ ID NO:19 anneals at the ligated linker andSEQ ID NO:20 anneals to the T-DNA sequence inside of the left border ofthe pRD29A-anti-FTA construct in Line 28.1. Two major bands wereproduced, one of 1.1 kb and the second of 1.2 kb. The reaction mixturefrom the first PCR amplification was diluted 1/50 and re-amplified usingnested primers identified by SEQ ID NO:21 and SEQ ID NO:22. Two majorDNA fragments were produced, one was about 1 kb and the second was 860bp. These final two DNA fragments were ligated into pBluescript T/Avector and sequenced.

Sequence analysis resulted in a match to a 860 bp fragment of theArabidopsis chromosome 1, BAC clone T23K23, Genbank accession numberAC012563. This region was identified and contained sequence from thecloned hydroxypyruvate reductase (HPR) coding sequence and upstreamsequence predicted to be a promoter. It was concluded that the T-DNA hadinserted into the promoter region of the HPR gene.

The insertion site was further characterized. Based on the genomicsequence data available from the Genbank database, the upstream regionwas determined to contain the gene phosphatidylinositol synthase (PIS1).Primers were produced, SEQ ID NO:23 and SEQ ID NO:26, and used toamplify portions of these genes and the intergenic region including theT-DNA insert. This primer set amplifies from within the PIS1 gene to aprimer within the NPTII gene contained on the T-DNA construct of line28.1. A larger than predicted fragment suggested that there was a tandeminsertion of the T-DNA. Subsequent PCR reactions to amplify the upstreamregion indicated that these T-DNA insertions were in oppositeorientations. Amplification of the upstream region was performed usingprimers identified by SEQ ID NO:23 and SEQ I) NO:20. Sequence analysisof the resulting PCR product contained the 3′ end of the PIS1 gene andthe intergenic region and matched that present in the data base fromGenbank Accession Number AC012563. The location of the T-DNA insertionwas thereby determined.

EXAMPLE 3 Cloning of Arabidopsis HPR cDNA and Promoter and VectorConstruction Using These Sequences

Using sequence data from the data base Accession Number AC012563,primers were designed to PCR amplify the HPR gene coding sequence. TotalRNA was isolated from Arabidopsis leaf tissue using the QIAGEN RNeasy®Plant kit.

The primer pair identified by SEQ ID NO:6 and SEQ ID NO:7 were used toamplify the appropriate gene sequence. The DNA fragment was cloned intopBluescript T/A vector at the EcoRV site and sequenced. To facilitatethe next cloning step the HPR gene was reamplified from the aboveplasmid using primer pairs SEQ ID NO:8 and SEQ ID NO:7. The resultingDNA fragment was digested with BamHI and cloned into pBI121, replacingthe BamHI fragment encoding the GUS gene. The resulting vector ispBI121-HPR.

The primer pair SEQ ID NO:9 and SEQ ID NO:10 was used to PCR amplify theRD29A promoter sequence which replaced the 35S promoter of plasmidpBI121-HPR to generate plasmid pRD29A-HPR.

Construction of the vector pBI121-hp-HPR, a hair pin RNAi construct, wasproduced as follows. The cloning strategy involved truncating the GUSgene of pBI121 and flanking the GUS sequence with a HPR fragment in theantisense orientation upstream of the GUS and in the sense orientationon the downstream side of GUS. The pBI121 vector was digested with SmaIand SacI, the GUS sequence and the vector fragments were purified fromone another. The isolated GUS fragment was digested using EcoRV and the1079 bp blunt ended EcoRV/SacI fragment isolated. This was ligated backinto the digested parent vector at the SmaI/SacI site. The HPR fragmentsto be cloned in opposite orientations were produced using the primerpairs identified by SEQ ID NO:8 and SEQ ID NO:11, to produce theantisense orientation and SEQ ID NO:12 and SEQ ID NO:13, to produce thesense orientation fragment. These primers incorporated restriction sitesadvantageous for the cloning strategy. The sense fragment was insertedat the SacI site and the antisense fragment between the XbaI and BamHIsites.

The HPR promoter was PCR amplified from genomic DNA isolated from wildtype Arabidopsis thaliana (ecotype Columbia) using primers which weredesigned based on sequence data in Accession number AC012563. The primerpair identified by SEQ D NO:14 and SEQ ID NO:15 were used to PCR amplifythe promoter and the first 2 codons of the HPR gene. The DNA fragmentwas cloned into pbluescript T/A vector at the EcoRV site and sequenced.The fragment was cloned into pBI121 at the HindIII and BamHI sites,replacing the 35S promoter of that plasmid. A truncated version of thepromoter was produced using the primer pair identified by SEQ ID NO:16and SEQ ID NO:15 and cloned as above. The resulting plasmids arereferred to as pHPR-GUS and pHPRT-GUS respectively.

EXAMPLE 4 Sequence Analysis

A disclosed nucleic acid of 1161 nucleotides (SEQ ID NO:1) and alsoreferred to as HPR, is shown in Table 1 TABLE 1A HPR Nucleotide Sequence(SEQ ID NO:1). ATGGCGAAACCGGTGTCCATTGAAGTGTATAATCCTAATGGGAAATACAGAGTTGTTAGCACAAAACCGATGCCTGGAACTCGCTGGATCAATCTCTTGGTAGACCAAGGTTGTCGCGTTGAGATATGTCATTTGAAGAAGACATCTTGTCTGTAGAAGATATCATTGATCTGATCGGAGACAAGTGTGATGGAGTCATCGGTCAGTTGACGGAAGATTGGGGAGAGACTCTGTTCTCAGCTTTGAGCAAAGCTGGAGGGAAAGCTTTCAGTAACATGGCCGTTGGTTATAACAACGTTGATGTTGAAGCTGCCAATAAGTATGGAATTGCTGTCGGTAACACTCCGGGAGTGTTGACTGAGACGACGGCTGAACTAGCTGCTTCTCTTTCCTTGGCTGCTGCAAGAGAAGAATTGTTGAAGCCGACGAATTCATGAGAGGTGGCTTGTACGAGGGATGGCTTCCTCATCTGTTTGTGGGGAACTTACTTAAAGGACAGACTGTTGGAGTTATTGGAGCTGGACGTATTGGATCTGCTTATGCTAGAATGATGGTGGAAGGGTTCAAGATGAATTTGATCTACTTTGATCTTTACCAATCCACTCGTCTTGAGAAATTTGTGACAGCTTATGGACAGTTCTTGAAAGCAAATGGAGAACAACCTGTGACATGGAAACGAGCTTCGTCCATGGAGGAGGTGCTGCGTGAGGCTGATCTGATAAGTCTTCACCCGGTGCTGGACAAAACCACTTACCATCTTGTCAACAAGGAGAGGCTTGCCATGATGAAAAAGGAAGCAATCCTTGTGAACTGCAGCAGAGGTCCTGTGATCGATGAGGCAGCTTTGGTCGAACATCTCAAAGAGAACCCGATGTTCCGAGTTGGTCTCGATGTGTTCGAGGAAGAGCCATTCATGAAACCAGGGCTTGCTGATACGAAAAACGCTATTGTTGTTCCTCACATTGCTTCTGCTTCCAAGTGGACTCGTGAAGGAATGGCTACGCTTGCAGCTCTCAACGTCCTCGGAAGAGTCAAAGGGTACCCGATTTGGCATGACCCGAACCGAGTCGATCCATTCTTGAACGAAAACGCTTCACCGCCCAATGCCAGTCCAAGCATCGTCAACTCAAAGGCCTTAGGATTGCCTGTT TCGAAGCTATGA

A disclosed HPR polypeptide (SEQ ID NO:2) encoded by SEQ ID NO:1 has 386amino acid residues and is presented in Table 1B using the one-letteramino acid code. TABLE 1B Encoded HPR protein sequence (SEQ ID NO:2).MAKPVSIEVYNPNGKYRVVSTKPMPGTRWINLLVDQGCRVEICHLKKTILSVEDIIDLIGDKCDGVIGQLTEDWGETLFSALSKAGGKAFSNMAVGYNNVDVEAANKYGIAVGNTPGVLTETTAELAASLSLAARRIVEADEFMRGGLYEGWLPHLFVGNLLGQTVGVIGAGRIGSAYARMMVEDGKMNLIYFDLYQSTRLEKFVTAYGQFLKANGEQPVTWKRASSMEEVLREADLISLHPVLDKTTYHLVNKERLAMMKKEAILVNCSRGPVIDEAALVEHLKENPMFRBGLDVFEEEPFMKPGLADTKNAIVVPHIASASKWTREGMATLAALNVLGRVKGYPIWHDPNRVDPFLNENASPPNASPSIVNSKALGLPVSKL

The present invention also includes a nucleic acid sequencecomplimentary to the Arabidopsis thaliana HPR of SEQ ID NO: 1. Thedisclosed complimentary sequence is shown as SEQ ID NO:3. TABLE 1CNucleotide Sequence Complimentary to HPR (SEQ ID NO:3).TCATAGCTTCGAAACAGGCAATCCTAAGGCCTTTGAGTTGACGATGCTTGGACTGGCATTGGGCGGTGAAGCGTTTTCGTTCAAGAATGGATCGACTCGGTTCGGGTCATGCCAAATCGGGTACCCTTTGACTCTTCCGAGGACGTTGAGAGCTGCAAGCGTAGCCATTCCTTCACGAGTCCACTTGGAAGCAGAAGCAATGTGAGGAACAACAATAGCGTTTTTCGTATCAGCAAGCCCTGGTTTCATGAATGGCTCTTCCTCGAACACATCGAGACCAACTCGGAACATCGGGTTCTCTTTGAGATGTTCGACCAAAGCTGCCTCATCGATCACAGGACCTCTGCTGCAGTTCACAAGGATTGCTTCCTTTTTTCATCATGGCAAGCCTCTCCTTGTTGACAAGATGGTAAGTGGTTTTGTCCAGCACCGGGTGAAGACTTATCAGATCAGCCTCACGCAGCACCTCCTCCATGGACGAAGCTCGTTTCCATGTCACAGGTTGTTCTCCATTTGCTTTCAAGAACTGTCCATAAGCTGTCACAAATTTCTCAAGACGAGTGGATTGGTAAGATCAAAGTAGATCAATTCATCTTGAACCCTTCCACCATCATTCTAGCATAAGCAGATCCAATACGTCCAGCTCCAATAACTCCAACAGTCTGTCCTTTAAGTAAGTTCCCCACAAACAGATGAGGAAGCCATCCCTCGTACAAGCCACCTCTCATGAATTCGTCGGCTTCAACAATTCTTCTTGCAGCAGCCAAGGAAAGAGAAGCAGCTAGTTCAGCCGTCGTCTCAGTCAACACTCCCGGAGTGTTACCGACAGCAATTCCATACTTATTGGCAGCTTCAACATCAACGTTGTTATAACCAACGGCCATGTTACTGAAAGCTTTCCCTCCAGCTTTGCTCAAAGCTGAGAACAGAGTCTCTCCCCAATCTTCCGTCAACTGACCGATGACTCCATCACACTTGTCTCCGATCAGATCAATGATATCTTCTACAGACAAGATTGTCTTCTTCAAATGACATATCTCAACGCGACAACCTTGGTCTACCAAGAGATTGATCCAGCGAGTTCCAGGCATCGGTTTTGTGCTAACAACTCTGTATTTCCCATTAGGATTATACACTTCAATGGACACCG GTTTCGCCATArabidopsis tiacliana HPR Promoter

A disclosed nucleic acid of 512 nucleotides (SEQ ID NO:4) and alsoreferred to as HPR promoter, is shown in Table 2A. TABLE 2A HPR PromoterSequence (SEQ ID NO:4).GAAGCAGCAGAAGCCTTGATCATCTTCCTTTGTCTCAACCTGAAACTCTTTTTTTTCTTTCATTGTTTGTTCTCTTTTCACTGTGGATGTAGATAATTGTTTTTAATGAAATGAAGAAATATTGATTTGCCTTTTGACATAATTTTGTTAATAATCTTGATTACAAATTTTAGTCAGTGTTTGATGCATAGTTGCATACTGCAGAGTTGAGTTTGGATATGGCCACGTCAGCATTATCTCGTTACCAAAACGTAAGGTCCAAACTCAGATAATACAAACGAAGCAGTTCTTTGTCACTCTATCATCAACATATGAACCACACCAAAAAAGAACAAAATCGTAGATAATGATCATGCAAAACCGACCGTTGGATCTTACTTTCGATTTCAAACCACATAAATCTTAGTGACTGAGCTAAAAAACTGAAATTTTTTAAAAGGCAAGACCTCCTCTGTTTCCATATTCTCACCACAGAAGAACTCTTGAGGCTTTCTCTTTTC TCTACCATGGCG

A disclosed nucleic acid of 288 nucleotides (SEQ ID NO:5) and alsoreferred to as HPR truncated promoter (HPRT), is shown in Table 2B.TABLE 2B HPR Truncated Promoter Sequence (SEQ ID NO:5).ACGTCAGCATTATCTCGTTACCAAAACGTAAGGTCCAAACTCAGATAATACAAACGAAGCAGTTCTTTGTCACTCTATCATCAACATATGAACCACACCAAAAAAGAACAAAATCGTAGATAATGATCATGCAAAACCGACCGTTGGATCTTACTTTCGATTTCAAACCACATAAATCTTAGTGACTGAGCTAAAAAACTGAAATTTTTTAAAAGGCAAGACCTCCTCTGTTTCCATATTCTCACCACAGAAGAACTCTTGAGGCTTTCTCTTTTCTCTACCATGGCG

EXAMPLE 5 Plant Transformation

Arabidopsis transgenic plants were made by the method of dippingflowering plants into an Agrobacterium culture, based on the method ofAndrew Bent in, Clough S J and Bent A F, 1998. Floral dipping: asimplified method for Agrobacterium-mediated transformation ofArabidopsis thaliana. Wild type plants were grown under standardconditions with a 16 hour, 8 hour light to dark day cycle, until theplant has both developing flowers and open flowers. The plant wasinverted for 2 minutes into a solution of Agrobacterium culture carryingthe appropriate gene construct. Plants were then left horizontal in atray and kept covered for two days to maintain humidity and then rightedand bagged to continue growth and seed development. Mature seed was bulkharvested.

Transformed T1 plants were selected by germination and growth on MSplates containing 50 μg/ml kanamycin. Green, kanamycin resistant(Kan^(R)) seedlings were identified after 2 weeks growth andtransplanted to soil. Plants were bagged to ensure self fertilizationand the T2 seed of each plant harvested separately. During growth of T1plants leaf samples were harvested, DNA extracted and Southern blot andPCR analysis performed.

T2 seeds were analyzed for Kan^(R) segregation. From those lines thatshowed a 3:1 resistant phenotype, surviving T2 plants were grown, baggedduring seed set, and T3 seed harvested from each line. T3 seed was againused for Kan^(R) segregation analysis and those lines showing 100%Kan^(R) phenotype were selected as homozygous lines. Further molecularand physiological analysis was done using T3 seedlings.

Transgenic Brassica napus, Glycine max and Zea maize plants can beproduced using Agrobacterium mediated transformation of cotyledonpetiole tissue. Seeds are sterilized as follows. Seeds are wetted with95% ethanol for a short period of time such as 15 seconds. Approximately30 ml of sterilizing solution I is added (70% Javex, 100 μl Tween20) andleft for approximately 15 minutes. Solution I is removed and replacedwith 30 ml of solution II (0.25% mecuric chloride, 100 μl Tween20) andincubated for about 10 minutes. Seeds are rinsed with at least 500 mldouble distilled sterile water and stored in a sterile dish. Seeds aregerminated on plates of ½ MS medium, pH 5.8, supplemented with 1%sucrose and 0.7% agar. Fully expanded cotyledons are harvested andplaced on Medium I (Nurashige minimal organics (MMO), 3% sucrose, 4.5mg/L benzyl adenine (BA), 0.7% phytoagar, pH5.8). An Agrobacteriumculture containing the nucleic acid construct of interest is grown for 2days in AB Minimal media. The cotyledon explants are dipped such thatonly the cut portion of the petiole is contacted by the Agrobacteriumsolution. The explants are then embedded in Medium I and maintained for5 days at 24° C., with 16,8 hr light dark cycles. Explants aretransferred to Medium II (Medium I, 300 mg/L timentin,) for a further 7days and then to Medium III (Medium II, 20 mg/L kanamycin). Any root orshoot tissue which has developed at this time is dissected away.Transfer explants to fresh plates of Medium III after 14-21 days. Whenregenerated shoot tissue develops the regenerated tissue is transferredto Medium IV (MMO, 3% sucrose, 1.0% phytoagar, 300 mg/L timentin, 20mg/L 20 mg/L kanamycin). Once healthy shoot tissue develops shoot tissuedissected from any callus tissue are dipped in 10×IBA and transferred toMedium V (Murashige and Skooge (MS), 3% sucrose, 0.2 mg/L indole butyricacid (D3A), 0.7% agar, 300 mg/L timentin, 20 mg/L 20 mg/L kanamycin) forrooting. Healthy plantlets are transferred to soil. The above method,with or without modifications, is suitable for the transformation ofnumerous plant species including Glycine max, Zea maize and cotton.

Transgenic Glycine max, Zea maize and cotton can be produced usingAgrobacterium-based methods which are known to one of skill in the art.Alternatively one can use a particle or non-particle biolisticbombardment transformation method. An example of non-particle biolistictransformation is given in U.S. Patent Application 20010026941. Thismethod has been used to produce transgenic Glycine max and Zea maizeplants. Viable plants are propagated and homozygous lines are generated.Plants are tested for the presence of drought tolerance, physiologicaland biochemical phenotypes as described elsewhere.

Transformation of plant tissue such as Zea maize for example, can beachieved by sonication of callus tissue culture. Callus tissue wasproduced as follows. Ears of corn were harvested 18 days after silkingand surface sterilized in 50% v/v bleach for 20 minutes followed bythree washing with sterile distilled water. Immature embryos ranging insize from 2 to 4 mm were harvested from the kernels. Embryos were placedon MSD_(1.5) medium (2% sucrose, 1×MS macronutrient and micronutrientsalts, 1×MS vitamins, 1.5 mg / L 2,4-D, 0.8% agar, pH 5.8) scutellumside up. Embryos were incubate at 26-28° C. in the dark. Friable callusfrom 2 week old cultures were transferred to fresh MSD_(1.5) medium andfurther incubated at 26-28° C. in the dark. Friable callus wassubcultured to fresh MSD_(1.5) medium every 21 days.

Transformation of callus tissue was performed as described below. Theconstruct was introduced into GV3101 Agrobacterium by inoculation of asingle colony of GV3101 Agrobacterium containing the HPR-GUS plasmidinto 10 mL of LB amended with 150 μg/mL rifampicin, 100 μg/mL gentamycinsulfate, and 50 μg/mL kanamycin. The culture was allowed to growovernight at 28° C. with 200 rpm shaklng. Corn callus was cut intopieces approximately 3-5 mm in size. The Agrobacterium culture wascentrifuged at 1500×g for 10 minutes and washed twice with 10 mL liquidMSD_(1.5) liquid (2% sucrose, 1×MS macronutrient and micronutrientsalts, 1×MS vitamins, 1.5 mg/L 2,4-D, pH 5.8). The bacteria wasresuspended in liquid MSD_(1.5) to an OD_(600 nm) of 0.25 and 1 mL ofdiluted Agrobacterium or liquid MSD_(1.5), for negative controls, wasplaced in 1.5 mL microfuge tubes containing four pieces of callus addedto each tube. Callus and Agrobacterium culture was sonicated in aBranson 200 Ultrasonic Cleaner for 0, 3, 10, 30, 100, or 300 secondswith bacteria or 0 or 300 seconds without bacteria (in MSD_(1.5) liquidalone). After sonication, the callus was blotted on sterile filter paperand placed on MSD_(1.5)A medium (MSD_(1.5) solid medium amended with 100μM acetosyringone). The co-cultivation period was 4 days in the dark at28° C. Callus was rinsed in liquid MSD_(1.5), blotted on sterile filterpaper, and placed on MSD_(1.5)T medium (MSD_(1.5) solid medium amendedwith 400 μg/mL Timentin) for 3 days in the dark at 28° C. Seven daysafter sonication, callus was added to 1 mL GUS staining solution (50 mMNaPO₄, pH 7.0, 0.1% Triton X-100, 1 mM EDTA, 2 mM DTT, 0.5 mg/mL X-GlcA)and left to incubate overnight at 37° C. The staining solution wasreplaced with 1 mL fixation buffer (10% formaldehyde, 50% ethanol) andincubated for 30 minutes at room temperature. The fixation buffer wasreplaced with 80% ethanol and incubated for 1 hour at room temperature.The 80% ethanol was replaced with 100% ethanol and incubated for 1 hourat room temperature. The callus was assessed for blue staining,indicating GUS activity.

EXAMPLE 6 Assessment of ABA Sensitivity

Approximately 100 seeds were assessed per line per 9 cm plate. Seedswere sterilized with 50% bleach for 8 minutes and washed four times withsterile water. Seeds were plated on minimal medium (½ MS salt, withoutsucrose and vitamins) supplemented with no ABA, 0.25 μM, 0.5 μM, or 1.0μM ABA. Plates were chilled for 3 days at 4° C. in the dark, andincubated for up to 21 days at 22° C. with 16 hour, 8 hour light to darklight cycle. Plates were assessed for germination, cotyledon expansion,true leaf development and seedling vigor. Seedlings were assessed forABA sensitivity over 21 days of growth at which time sensitive seedlingswere arrested at the cotyledon stage, lacked true leaves, and showedinhibition of root growth. Wild type control Columbia plants had two tothree pairs of true leaves and a well developed root system. Lines werecategorized as ABA sensitive (ABA^(S)) if less than 1% of plants lookedlike control, moderately ABA sensitive (ABA^(MS)) if more than 1% butless than 50% of looked like control, or ABA insensitive (ABA^(Wt)) ifgreater than 50% looked like control. For example, if a plate had 20healthy seedlings and the control plate had 60 healthy seedlings, theline would be 33% of control and categorized as moderately ABAsensitive.

All three vector constructs (pRD29A-HPR, pBI121-HPR AND pBI121-hp-HPR)have resulted in transgenic lines of Arabidopsis which have increasedsensitivity to ABA which is indicative of stress tolerance. Nineteenlines of pRD29A-HPR were ABA^(S) at 0.5 μM ABA, seven pBI121-HPR linesand three pBI121-hp-HPR lines showed ABA^(S) characteristics.

EXAMPLE 7 Southern Blot Analysis

Genomic Southern blot analysis of transgenic Arabidopsis was performedusing standard techniques known to one skilled in the art. Typically, 5μg of DNA was electrophoresed in a 0.8% agarose gel and transferred toan appropriate membrane such as Hybond N+ (Amersham Pharmacia Biotech).Pre-hybridization and hybridization conditions were as suggested by themembrane manufacturer, typically at 65° C. The final stringency wash wastypically at 1×SSC and 0.1% SDS at 65° C. The NPTII coding region wastypically used as the radiolabeled probe in Southern blot analysis.Transgenic Arabidopsis lines were selected as homozygous based onsegregation patterns observed from NPTII probed Southern blots. Lineswere confirmed to be transgenic by PCR analysis using transgene specificprimers in the PCR assays. Lines of pBI121-HPR were confirmed using theprimer pair identified by SEQ ID NO:27 and SEQ ID NO:7. Lines ofpRD29A-HPR were confirmed using the primer pair identified by SEQ IDNO:9 and SEQ ID NO:7. Lines of pBI121-hp-HPR were confirmed using theprimer pair identified by SEQ ID NO:27 and SEQ ID NO:8.

EXAMPLE 8 Northern Blot Analysis

Total RNA was isolated from transgenic and wild type Arabidopsis lines(T3 plants). Approximately 10 μg of total RNA was loaded into each lane.The Northern was probed with radiolabeled HPR cDNA in ExpressHybhybridization solution (Clontech) and exposed using a phosphoimagingscreen. For quantification blots were reprobed with tubulin, aconstitutively expressed gene, for a comparative standard.

A survey of HPR expression in various tissues of wild type plants showsthat expression is detectable in aerial tissues but not in roots.

The endogenous HPR expression level in wild-type Arabidopsis leavesduring drought stress treatment was examined. Plants were grown underoptimal conditions in the growth chamber for three weeks beforewithholding water to mimic a drought stress. Leaf samples were collectedfor total RNA extraction at day 0, 2, 4, and 6 after cessation ofwatering. The level of HPR gene expression increased over the waterstress, to a maximum expression level, which was 60% greater thaninitial, as of day 4 of the drought stress. This demonstrates theup-regulation of Arabidopsis HPR expression by an abiotic stress such asdrought.

Northern analysis shows that the HPR expression found in Line 28.1 isapproximately half of that found in wild type plants. Thereby providingfurther data that HPR is the affected gene in Line 28.1 PIS1 expressionwas the same in line 28.1 as in the wild type control.

Transgenic pBI121-HPR lines were assessed for HPR expression by Northernanalysis. Thirteen of eighteen lines examined showed an increase in geneexpression, typically by two or three fold but also to as much asfourteen fold increase. The lines that showed altered HPR expressioncorrelated with the lines that showed increased ABA sensitivity.

Transgenic pBI121-hp-HPR lines were assessed for HPR expression byNorthern analysis. Seven lines examined showed a decrease in geneexpression with two lines having undetectable expression. The lines thatshowed altered HPR expression correlated with the lines that showedincreased ABA sensitivity.

Transgenic pRD29A-HPR lines are assessed for HPR expression by Northernanalysis. The pRD29A promoter is a drought inducible promoter and assuch Northern analysis is conducted after plants have been exposed to adrought stress treatment.

EXAMPLE 9 HPR Promoter Analysis

Transgenic Arabidopsis lines of pHPR-GUS and pHPRT-GUS were analyzed forGUS staining activity to determine the expression characteristics of theisolated promoter fragments. One week old seedlings were incubated at37° C. in staining buffer (50 mM NaPO₄ pH 7.0, 10 mM EDTA, 0.1% TritonX-100, 0.5 mg/mL X-Gluc) for 16 hours. Wild type control plants showedno GUS staining while transgenic plants having the pHPR-GUS constructshowed staining in aerial tissue of young seedlings (FIG. 3B) but nodetectable expression in roots (FIG. 3C). Analysis of flowers showed GUSactivity particularly in the petals and stigma tissue. Comparison ofpHPR-GUS transgenic plants to those harboring the p121-GUS constructcontaining the 35S CaMV promoter shows that the intensity of expressionby the HPR promoter is equivalent to that of the 35S promoter (FIGS.3A,B).

Analysis of pHPRT-GUS lines did show expression in the roots in additionto the staining of the aerial tissues (FIG. 3C). The strength and aerialexpression patterns of the pHPRT-GUS construct is equivalent to thepHPR-GUS construct.

The pHPR-GUS and pHPRT-GUS promoters were evaluated in Brassica napus(canola) transgenic plants. The patterns observed in Arabidopsis wereagain seen in the canola plants (FIG. 4A). Analysis of the siliques anddeveloping seeds showed expression in these tissues (FIG. 4B).

The pHPR-GUS promoter was evaluated in Zea maize (corn) transgeniccallus tissue. The construct produced GUS activity in transformed corncallus tissue (FIGS. 5, 1A,1B). Negative controls did not produce GUSactivity.

EXAMPLE 10 HPR Activity Assay

HPR enzymatic activity was assessed using a biochemical assay, anexample of such an assay is as follows. Leaf tissue was ground inextraction buffer (25 mM Tris pH 7.5, 0.2 mM EDTA, 0.5 mM PMSF, 0.5μg/mL leupeptin, 0.2 μg/mL pepstain A. 2 mM DTT, 1% Tritin X-100). An 19μL aliquot of the clarified supernatant solution was added; to 196 μL ofthe assay solution (10 mM NADH, 150 mM hydroxypyruvate, 50 mM Tris pH7.5). The change in absorbance was monitored spectophotometrically at340 nm.

EXAMPLE 11 Production of Polyclonal Antibodies Against HPR

Anti-AtHPR antibodies were generated using AtHPR fusion proteinover-expressed in E. coli. The over-expression vector, pMAL-p2, contains1175 bp malE gene that is located upstream of AtHPR and encodes a 43 KDamaltose-binding protein (MBP). The AtHPR fragment to be used was PCRamplified using primer pair identified by SEQ ID NO:28 and SEQ ID NO:29.The 1161 bp XbaII/SalI DNA fragment of AtHPR was inserted into pMAL-p2at XbaII and SalI sites. The SalI site was converted into blunt endusing Klenow fragment. The resulting fusion protein MBP-AtHPR was thenover-expressed in DH5α, and purified by one-step annnity for MBP asdescribed by the manufacturer (New England Biolab). The soluble fractionof the crude bacterial extract containing the MBP-AtHPR fusion proteinwas loaded to a amylose column (1.5 cm×10.0 cm), and the proteins wereeluted with 10 mM maltose in column buffer (50 mM Tris-HCl, pH 7.5, 1 mMEDTA, and 200 mM NaCl). Fractions containing purified MBP-AtHPR fusionprotein were pooled, and concentrated with a Centriprep-30 concentrator(Amicon). All purification steps were carried out at 4° C. To generatean antibody, the purified fusion protein was further separated bySDS-PAGE and the Coomassie stained band corresponding to the fusionprotein was excised. The identity of the fusion protein was confirmed byWestern analysis using anti-MBP antibodies (purchased from New EnglandBiolab). The protein was eluted from the gel slice by electroelution andthen emulsified in Ribi adjuvant (Ribi Immunochem) to a final volume of1 ml. MBP-AtHPR protein was injected into a 3 kg New Zealand rabbit onday 1 and booster injections were given on day 21 and day 35 with 250 μgof the protein each time. High-titer antisera were obtained one weekafter the final injection.

EXAMPLE 12 Western Blot Analysis of HPR Transgenic Lines UsingAnti-AtHPR Antibodies

Western analysis was performed to examine expression level of AtHPR inthe transgenic lines compared with that of wild type plants. Anti-Bipantibody, an ER lumenal protein (Stressgen, Victoria, BC, Canada) wasused as a reference. Total proteins were extracted from developing leaftissue of ABA^(S) lines and a wild type control. The antigenic proteinbands of AtHPR and Bip were scanned and quantified using the UN-Scan-Itprogram (Silk Scientific, Utah, USA) and the ratio of the two proteinbands for each sample was obtained.

EXAMPLE 13 Physiology Analysis, Drought

Transgenic lines of Arabidopsis plants carrying the pBI121-HPR andpRD29A-HPR constructs were analyzed for drought tolerant phenotypecharacteristics. Performance of the transgenic lines was compared tothat of Columbia wild type controls.

Seeds were germinated on agar plates containing ½ MS, 1% sucrose, 5 mMMES and vitamins. After one week, seedlings were transplanted into 3″pots, five seedlings per pot, with consistent amounts of soil. Sixreplicate pots were planted per line. Plants were grown under optimalconditions (16 hr light at 200 uE, 70% RH, and 22C), watered daily untilthe first open flower. The drought treatment was initiated by wateringup the pots to equal pot weight followed by cessation of furtherwatering. Pots were weighed daily for four days at which time rosetteleaves, and stems were harvested for fresh and dry weightdeterminations. Daily soil water content, and final plant turgidity(ratios of fresh to dry weight) were calculated. In addition, water lostin first two days/final leaf dry biomass and water lost in 2 days/finaltotal shoot dry biomass were determined.

Drought tolerant lines were identified as lines which had greater soilwater content, lower water loss per dry biomass and equal or greater drybiomass. They visually looked more turgid than controls and thisobservation was confirmed by greater fresh to dry weight ratios.

Transgenic lines of pBI121-HPR were advanced to the T3 generation andconfirmed to be homozygous. Lines were assessed for days to firstflower, water loss per day as a percentage of initial, water loss aftertwo days drought treatment relative to biomass and fresh and dry weightsof leaves and stems. Five lines were identified that demonstrated adrought tolerant phenotype.

Seven lines had significantly higher soil water content but three ofthose lines also had reduced biomass. Water lost in 2d/g of leaf dryweight was significantly greater for eight of the lines eliminatingthese as drought tolerant candidates. Three lines had significantlyreduced water loss/g leaf dry weight compare to Columbia. Water lost in2 d/g shoot dry weight was significantly greater for six lines and nolines had significantly lower water loss/shoot DW. Four of the lines hadsignificantly higher rosette leaf DW and most of the other lines weresignificantly reduced in leaf DW as compared to Columbia. Stem DW wassignificantly reduced in four of the lines and the rest of the lineswere not different from Columbia. Therefore the overall shoot DW wassignificantly reduced in ten lines. There were a number of significantdifferences in FW/DW ratios for both, stem and leaf as well as shoot butthese were a reflection of smaller plants using less water. Five of thelines flowered significantly earlier, by up to two days earlier, thanColumbia.

Transgenic lines of pRD29A-HPR were advanced to the T3 generation andconfirmed to be homozygous. Lines were assessed for days to firstflower, water loss per day as a percentage of initial, water loss aftertwo days drought treatment relative to biomass and fresh and dry weightsof leaves and stems. Eighteen lines were identified that demonstrated adrought tolerant phenotype.

Eight lines showed delayed flowering by up to two days. Most of thelines showed significantly increased soil water content, and all exceptone line showed significantly reduced water lost in 2 d/g leaf dryweight. Water lost in 2 d/g shoot dry weight was also significantlyreduced in the majority of lines. All lines, except one, hadsignificantly reduced stem dry weight and significantly increasedrosette leaf dry weight. Additionally, stem, leaf and shoot fresh to dryweight ratios were significantly increased in many lines indicatinghigher water content and turgidity of these lines.

Transgenic lines of pBI121-hp-HPR were advanced to the T3 generation andconfirmed to be homozygous. Lines are assessed for days to first flower,water loss per day as a percentage of initial, water loss after two daysdrought treatment relative to biomass and fresh and dry weights ofleaves and stems.

EXAMPLE 14 Physiology Analysis, Photo-Oxidative Stress

Growth of transgenic lines of pBI121-hp-HPR using a 24 hour light regimeresulted in plants that were under visible stress. Leaves were chloroticand plants were generally smaller and less vigorous than wild typecontrols. Growth of these plants using a 16 hour light, 8 hour darkcycle relieved the symptoms of stress and plants appeared to grownormally.

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1. An isolated nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:4 or SEQ ID NO: 5 wherein said nucleic acid molecule isless than 1000 nucleotides in length.
 2. The isolated nucleic acidmolecule of claim 1, wherein said nucleic acid molecule is less than 800nucleotides in length.
 3. The isolated nucleic acid molecule of claim 1,wherein said nucleic acid molecule is less than 750 nucleotides inlength.
 4. The isolated nucleic acid molecule of claim 1, wherein saidnucleic acid molecule is less than 600 nucleotides in length.
 5. Anisolated nucleic acid molecule consisting of the nucleotide sequence ofSEQ ID NO:4 or fragment thereof.
 6. An isolated nucleic acid moleculeconsisting of the nucleotide sequence of SEQ ID NO:5 or fragmentthereof.
 7. The isolated nucleic acid molecule of claim 1, wherein saidsequence regulates transcription of an operably linked nucleotidesequence of interest.
 8. The isolated nucleic acid molecule of claim 7,wherein said sequence regulates transcription by inducing expression inresponse to a stimulus.
 9. The isolated nucleic acid molecule of claim8, wherein said stimulus is light or an environmental stress.
 10. Aisolated nucleic acid construct comprising, a promoter sequencecomprising the nucleic acid sequence of SEQ ID NO: 4 or 5 or fragmentthereof operably linked to a nucleotide sequence encoding a heterologousgene, wherein said heterologous gene encodes a protein of interest orfragment thereof.
 11. The construct of claim 10, wherein said constructcomprises at least two promoter sequences.
 12. The construct of claim11, further comprising a spacer sequence, wherein said spacer sequenceoperably links said promoter sequences.
 13. The construct of claim 10,further comprising a nucleic acid encoding a selectable marker.
 14. Theconstruct of claim 10, further comprising a nucleic acid encoding areporter gene.
 15. The construct of claim 10, wherein said heterologousgene is capable of altering an agronomic trait.
 16. The construct ifclaim 15, wherein said agronomic trait is disease resistance, herbicideresistance, environmental stress resistance, enhanced growth, orincreased yield.
 17. The construct of claim 10, wherein saidheterologous gene is a plant gene.
 18. The construct of claim 10,wherein said heterologous gene is a structural gene.
 19. The constructof claim 18, wherein said structural gene is an enzyme, atranscriptional regulator, a chaperonin protein or a scaffoldingprotein.
 20. The construct of claim 19, wherein said enzyme is farnesyltransferase alpha, farnesyl transferase beta or CaaX prenyl protease.21. A isolated nucleic acid construct comprising, a promoter sequencecomprising SEQ ID NO: 4 or 5 or fragment thereof operably linked to anon-translatable mRNA molecule of a gene encoding a protein of interest.22. The construct of claim 21, wherein said non-translated mRNA moleculeis an antisense nucleic acid, a hairpin RNA or a microRNA.
 23. Theconstruct of claim 21, fuirther comprising a nucleic acid encoding aselectable marker.
 24. The construct of claim 21, further comprising anucleic acid encoding a reporter gene.
 25. The construct of claim 21,wherein said gene is capable of altering an agronomic trait.
 26. Theconstruct if claim 25, wherein said agronomic trait is diseaseresistance, herbicide resistance, environmental stress resistance,enhanced growth or increased yield.
 27. The construct of claim 21,wherein said gene is a plant gene.
 28. The construct of claim 21,wherein said gene is a structural gene.
 29. The construct of claim 28,wherein said structural gene is an enzyme, a transcriptional regulator,a chaperonin protein or a scaffolding protein.
 30. The construct ofclaim 29, wherein said enzyme is farnesyl transferase alpha, farnesyltransferase beta or CaaX prenyl protease.
 31. A vector comprising thenucleic acid molecule of claim
 1. 32. A cell comprising the vector ofclaim
 31. 33. The cell of claim 32, wherein said cell is a plant cell.34. The cell of claim 33, wherein said plant cell is monocotyledonous.35. The cell of claim 33, wherein said plant cell is dicotyledonous. 36.A vector comprising the nucleic acid construct of claim
 10. 37. A cellcomprising the vector of claim
 36. 38. The cell of claim 37, whereinsaid cell is a plant cell.
 39. The cell of claim 38, wherein said plantcell monocotyledonous.
 40. The cell of claim 38, wherein said plant cellis dicotyledonous.
 41. A vector comprising the nucleic acid construct ofclaim
 21. 42. A cell comprising the vector of claim
 41. 43. The cell ofclaim 42, wherein said cell is a plant cell.
 44. The cell of claim 43,wherein said plant cell monocotyledonous.
 45. The cell of claim 43,wherein said plant cell is dicotyledonous.
 46. A method of producing atransgenic plant comprising introducing into a plant cell the vector ofclaim 36, to generate a transgenic cell and regenerating a transgenicplant from said transgenic cell, wherein said transgenic plant expressessaid protein of interest.
 47. The method of claim 46, wherein saidexpression is constitutive.
 48. The method of claim 46, wherein saidexpression is inducible
 49. The method of claim 46, wherein said plantcell is monocotyledonous.
 50. The method of claim 46, wherein said plantcell is dicotyledonous.
 51. A method of producing a transgenic plantcomprising introducing into a plant cell the vector of claim 41, togenerate a transgenic cell and regenerating a transgenic plant from saidtransgenic cell, wherein said transgenic plant expresses said protein ofinterest at a decreased level as compared to a wildtype plant
 52. Themethod of claim 51, wherein said plant cell is monocotyledonous.
 53. Themethod of claim 51, wherein said plant cell is dicotyledonous.
 54. Thetransgenic plant produced by the method of claim
 46. 55. The seedproduced by the transgenic plant of claim 54, wherein said seed producesa plant that expresses said protein of interest.
 56. The transgenicplant produced by the method of claim
 51. 57. The seed produced by thetransgenic plant of claim 56, wherein said seed produces a plant thatexpresses said protein of interest at a decreased level as compared to awildtype plant.
 58. A method of expressing a heterologous proteincomprising introducing to a cell the construct of of claim 10 andexpressing said heterologous protein in said cell.
 59. The method ofclaim 58, wherein said cell is a plant cell.
 60. The method of claim 59,wherein said plant cell is monocotyledonous.
 61. The method of claim 59,wherein said plant cell is dicotyledonous.